Anti-pcsk9-glp-1 fusions and methods for use

ABSTRACT

This application provides anti-PCSK9˜GLP-1 fusions and methods for use.

FIELD

Anti-PCSK9˜GLP-1 Fusions and Methods for Use

BACKGROUND

Diabetes is associated with higher cardiovascular morbidity and mortality. Hypertension, hyperlipidemia, and diabetes are independently associated with increased risk of cardiovascular disease. Subjects with Type 2 diabetes are at two- to four-fold increased risk of cardiovascular disease compared to those without diabetes.

Glucagon-like peptide-1 (GLP-1) is known as a pleiotropic peptide with metabolic and cardiovascular benefits. It is derived from pre-proglucagon, a 158 amino acid precursor polypeptide that is processed in different tissues to form a number of different proglucagon-derived peptides, including glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin (OXM), that are involved in a wide variety of physiological functions, including glucose homeostasis, insulin secretion, gastric emptying, and intestinal growth, as well as the regulation of food intake. GLP-1 is produced as a 37-amino acid peptide that corresponds to amino acids 72 through 108 of proglucagon (92 to 128 of preproglucagon). The predominant biologically active form is a 30-amino acid peptide hormone (GLP-1(7-37) acid) that is produced in the gut following a meal and rapidly degraded by an abundant endogenous protease-DPP4. Baggio, L. and Drucker, D., Gasteroenterology, 132:2131-2157 (2007).

GLP-1 and GLP-1 analogs, acting as agonists at the GLP-1 receptor, have been shown to be effective hypoglycemic control, e.g., type-2 diabetes. Certain GLP-1 analogs are being sold or are in development for treatment of type-2 diabetes including, e.g., liraglutide (Victoza® from Novo Nordisk), dulaglutide (Eli Lilly), Bydureon (AZ/BMS), Aliblutide (GSK) and Exenatide (Byetta® from Eli Lilly/Amylin).

One of the primary side effects following the initiation of GLP-1 therapy is gastrointestinal side effects, particularly nausea. This side effect is transient, resolves over time and can be mitigated by dose escalation. However, therapy is limited to patients that can tolerate the gastrointestinal side effects.

PCSK9 is a nonenzymatic target for LDL cholesterol reduction and PCSK9 mutations correlate with reductions in LDL cholesterol and coronary heart disease. Cohen J C, N Engl J Med, 354:1264 (2006). PCSK9 antibodies have been shown to reduce LDL cholesterol in statin-treated patients and multiple candidates are undergoing clinical review.

While there are a plurality of individual treatments for diabetes and cardiovascular diseases, there is a need for a single pharmaceutical composition to address both disease states (and the relationship between diabetes and cardiovascular disease). Providing a single pharmaceutical compound that has dual activities will reduce side effects, difficulties with patient compliance, and will increase beneficial outcomes to individual patients and will decrease costs incurred by the health care system.

SUMMARY

In accordance with the description, disclosed is a dual active fusion molecule for the treatment of diabetes comprising an anti-PCSK9 antibody stably fused to a GLP-1 peptide, wherein the anti-PCSK9 antibody binds a PCSK9 polypeptide and the GLP-1 peptide binds a GLP-1 receptor.

In one aspect, wherein the GLP-1 peptide is fused to the PCSK9 antibody via a linker peptide.

In a further mode, the linker peptide is fused to the C-terminus of the GLP-1 peptide.

In one embodiment, the GLP-1 peptide comprises the amino acid sequence of SEQ ID NO: 36.

In one embodiment, the GLP-1 peptide comprises the amino acid sequence of SEQ ID NO: 3.

In one mode, wherein the Cys18 of the GLP-1 molecule forms a disulfide bridge with the linker peptide or with the GLP-1 peptide itself.

In one aspect, the fusion molecule controls glucose and/or reduces LDL in an animal. The animal may be human.

Disclosed also is a dual active fusion molecule for the treatment of diabetes comprising an anti-PCSK9 antibody stably fused to a GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 3, wherein C-terminus of the GLP-1 peptide is fused via a peptide linker to the light chain of the anti-PCSK9 antibody, and wherein the anti-PCSK9 antibody binds a PCSK9 polypeptide and the GLP-1 peptide binds a GLP-1 receptor.

Another embodiment disclosed is a method of treating Type 2 Diabetes comprising administering to a subject in need thereof, a fusion molecule described herein.

A further aspect comprises administering to a subject in need thereof, a fusion molecule described herein.

In one mode, a method of reducing low density lipoprotein (LDL) in a subject comprises administering to a subject in need thereof, a fusion molecule described herein.

Another aspect encompasses a method of controlling glucose and reducing LDL in a subject comprising administering to a subject in need thereof, a fusion molecule described herein.

Another aspect encompasses a method of promoting weight loss and reducing LDL in a subject comprising administering to a subject in need thereof, a fusion molecule described herein.

In one embodiment, the subject has Type 2 diabetes.

In another embodiment, the subject has metabolic syndrome.

An additional aspect is a dual active fusion molecule comprising an antibody stably fused to a GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 3, wherein C-terminus of the GLP-1 peptide is fused via a peptide linker to the light chain of the antibody, and wherein the antibody binds a target polypeptide and the GLP-1 peptide binds a GLP-1 receptor.

A further aspect is a dual active fusion molecule comprising an antibody stably fused to a GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 36, wherein C-terminus of the GLP-1 peptide is fused via a peptide linker to the light chain of the antibody, and wherein the antibody binds a target polypeptide and the GLP-1 peptide binds a GLP-1 receptor.

In some aspects, the GLP-1 molecule comprises the amino acid sequence of SEQ ID NO: 36. In some aspects, the antibody is an anti-PCSK9 antibody.

In certain modes, the light chain of the anti-PCSK9 antibody is at least 90% identical to the amino acid sequence of SEQ ID NO: 2. In other modes, the light chain of the anti-PCSK9 antibody comprises the amino acid sequence of SEQ ID NO: 2. In yet further modes, the heavy chain of the anti-PCSK9 antibody is at least 90% identical to the amino acid sequence of SEQ ID NO: 1. In some aspects, the heavy chain of the anti-PCSK9 antibody comprises the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments of the dual active fusion molecule, the Cys18 of the GLP-1 molecule forms a disulfide bridge with the C terminus of the GLP-1 peptide.

In some embodiments, the dual active fusion molecule is for the treatment of diabetes. In some embodiments, the dual active fusion molecule controls glucose and/or reduces LDL in an animal.

In some modes, the animal is a human.

Some embodiments include a dual active fusion molecule for the treatment of diabetes comprising an anti-PCSK9 antibody stably fused to a GLP-1 peptide that has reduced potency at the human GLP-1 receptor compared to a GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 29, wherein the C-terminus of the GLP-1 peptide is fused via a peptide linker to the anti-PCSK9 antibody, and wherein the anti-PCSK9 antibody binds a PCSK9 polypeptide and the GLP-1 peptide binds a GLP-1 receptor. In some embodiments, the GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29 is fused using a linker comprising SEQ ID NO:4 to the amino acid sequence comprising SEQ ID NO: 416. In further embodiments, the dual active fusion molecule of claim 25 or 26, wherein the potency at the human GLP-1 receptor is reduced by 30 to 60 fold compared to a GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29.

Some embodiments include an isolated polynucleotide encoding the fusion molecule described herein.

In certain modes, encompassed is a vector comprising the polynucleotide described herein. In other modes, a host cell comprises the polynucleotide or vector described herein.

In some embodiments, a method of making the fusion molecule comprises culturing the host cell under conditions allowing expression of the fusion molecule, and recovering the fusion molecule.

In some modes, a pharmaceutical composition comprises the fusion molecule described herein and a carrier. In some modes, a kit comprises the composition described herein.

Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic for a dual action fusion molecule as described herein. FIGS. 1B-D show various schematics of certain embodiments of antibody-peptide fusion molecules.

FIG. 1E shows alignment using numerical numbering of PC9#2 variable heavy chain with germline sequence 1-46 (DP-7). Discrepancies are shown in white with a black background.

FIG. 1F shows alignment using numerical numbering of PC9#2 variable light chain with germline sequence VK1 O18 O8 (DPKI). Discrepancies are shown in white with a black background.

FIGS. 2A-G show inhibition of human PCSK9 binding to anti-PCSK9 mAbs by anti-PCSK9 antibody/GLP-1 peptide fusions at the heavy chain N-terminus.

FIGS. 3A-G show inhibition of human PCSK9 binding to anti-PCSK9 mAbs by anti-PCSK9 antibody/GLP-1 peptide fusions at the light chain N-terminus.

FIGS. 4A-B show activation of human GLP1-Receptor by anti-PCSK9 antibody/GLP-1 peptide N-terminus fusions at the antibody heavy chain (A) and light chain (B).

FIG. 5A illustrates stability in rat for a GLP-1 analogue in heavy chain fusion with the control antibody NIP228.

FIG. 5B demonstrates stability in rat for an Exendin-4 GLP-1 analogue in light chain fusion with the anti-PCSK9 antibody PC9#2.

FIG. 5C demonstrates stability in mice for a GLP-1 analogue in fusion with human IgG4 Fc fragment.

FIG. 5D demonstrates stability in mice for a GLP-1 analogue in light chain fusion with the anti-PCSK9 antibody PC9#2.

FIG. 6 demonstrates a potential target profile to guide PCSK9 affinity and GLP-1 potency.

FIG. 7A provides peptide and linker amino acid sequences for eight compounds with an incorporated N-glycosylation consensus motif.

FIG. 7B provides peptide amino acid sequence for three compounds incorporating a disulphide bridge.

FIG. 8 shows a visual representation of the PC9#2_GLP1 molecule and the anti-PC9#2 antibody and GLP-1Fc(G4) used as a benchmark control.

FIG. 9 shows stability in rat of NIP228_GLP1_VH, a GLP-1 analogue in heavy chain fusion with the control antibody NIP228).

FIG. 10 shows stability in mice of PC9#2_GLP-1_VL, a GLP-1 analogue in light chain fusion with the anti-PCSK9 antibody PC9#2.

FIG. 11 shows stability in mice of PC9#2_DSB#3, Exendin-4 analogue DSB#3 in light chain fusion with the anti-PCSK9 antibody PC9#2.

FIG. 12 shows stability in mice of PC9#2_NGS#7, GLP-1 analogue NGS#7 in light chain fusion with the anti-PCSK9 antibody PC9#2.

FIG. 13A illustrates stability in mice for GLP-1 analogue NGS#7 in light chain fusion with the anti-PCSK9 antibody PC9#2.

FIG. 13B shows stability in mice for Exendin-4 analogue DSB#1 in light chain fusion with the anti-PCSK9 antibody PC9#2.

FIG. 13C illustrates stability in mice for Exendin-4 analogue DSB#3 in light chain fusion with the anti-PCSK9 antibody PC9#2.

FIG. 14A shows stability in mice for benchmark compound GLP-1-Fc fusion Open squares: concentration of test molecule in serum over time; open diamonds concentration of “active” test molecule (as measured by GLP-1 activity) over time for the same samples.

FIG. 14B shows stability in mice for Exendin-4 analogue DSB#1 in light chain fusion with the anti-PCSK9 antibody PC9#2. Open squares: concentration of test molecule in serum over time; open diamonds concentration of “active” test molecule (as measured by GLP-1 activity) over time for the same samples.

FIG. 15A provides a preparative SEC chromatogram for Exendin-4 analogue DSB#1 in light chain fusion with the anti-PCSK9 antibody PC9#2 after initial protein A purification.

FIG. 15B provides a preparative SEC chromatogram for Exendin-4 analogue DSB#3 in light chain fusion with the anti-PCSK9 antibody PC9#2 after initial protein A purification.

FIG. 16 shows an analytical SEC-HPLC profile of Exendin-4 analogue DSB#1 in light chain fusion with the anti-PCSK9 antibody PC9#2 after initial protein A purification.

FIG. 17 shows the amino acid sequence of additional Exendin-4 variant peptides incorporating a cysteine bridge. Cysteine residues are shown in black, other mutated residues are shown as underline and additional glycine residues at the C-terminus cap are shown in grey.

FIG. 18 provides a preparative SEC chromatogram for Exendin-4 analogue DSB#7 in light chain fusion with the anti-PCSK9 antibody PC9#2 after initial protein A purification.

FIG. 19 provides a preparative SEC chromatogram for Exendin-4 analogue DSB#9 in light chain fusion with the anti-PCSK9 antibody PC9#2 after initial protein A purification.

FIG. 20 shows stability in rat for Exendin-4 analogue DSB#7 in light chain fusion with the anti-PCSK9 antibody PC9#2.

FIG. 21 illustrates stability in rat for Exendin-4 analogue DSB#9 in light chain fusion with the anti-PCSK9 antibody PC9#2.

FIG. 22A shows simulation in human of the impact PCSK9/GLP-1 fusion for PCSK9 suppression.

FIG. 22B illustrates simulation in human of PCSK9/GLP-1 fusion molecule for GLP-1 agonism activity compared to Dulaglutide.

FIG. 23 shows the improved monomeric profile for PC9_2_DSB#7.

FIG. 24 illustrates the pharmacokinetic profile of peptide/antibody molecules in rats after a single i.v. injection.

FIG. 25 demonstrates inhibition of human PCSK9 binding to LDL receptor using competition ELISA.

FIG. 26 shows inhibition of human PCSK9-dependent loss of LDL uptake in HepG2 cells.

FIG. 27 shows the engineered reduction of potency at the human GLP-1 receptor.

FIG. 28A show a human GLP1r cAMP assay for Exendin-4 GLP-1 analogue DSB7_V19A in light chain fusion with the anti-PCSK9 antibody HS9.

FIG. 28B illustrates the stability in rat for Exendin-4 GLP-1 analogue DSB7_V19A in light chain fusion with the anti-PCSK9 antibody HS9.

FIGS. 29A-D show specificity of the fusion molecule HS9_DSB7_V19 for GLP-1 receptor determined by cAMP assay.

FIGS. 30A-B show superior glucose control (A) and weight loss (B) over time, including at day 7 post dose.

FIG. 31 shows binding to human PCSK9 of HS9 anti-PCSK9 antibody in fusion with GLP-1 analogue peptide DSB7_V19A using different linkers.

FIG. 32 shows human GLP-1 receptor activation using GLP-1 analogue peptide DSB7_V19A in fusion with anti-PCSK9 antibody HS9 using different linkers.

FIG. 33 illustrates binding to human PCSK9 of anti-PCSK9 antibodies in fusion with GLP-1 analogue peptide DSB7_V19A

FIG. 34 illustrates human GLP-1 receptor activation using GLP-1 analogue peptide DSB7_V19A in fusion with anti-PCSK9 antibodies

FIG. 35 demonstrates binding to human B7-H1 of anti-B7-H1 antibody 2.7A4 in fusion with GLP-1 analogue peptide DSB7_V19A

FIG. 36 shows human GLP-1 receptor activation using GLP-1 analogue peptide DSB7_V19A in fusion with anti-B7-H1 antibody 2.7A4.

FIG. 37 describes stability in rat for the fusion molecule HS9_DSB7_V19A following a single i.v. injection at 60, 30 or 10 mg/kg.

FIG. 38 shows free PCSK9 concentration in rat following a single i.v. injection at 60, 30, or 10 mg/kg of HS9_DSB7_V19A.

FIG. 39 shows total compound, active compound at GLP-1 receptor and free PCSK9 concentrations in rat following a single subcutaneous injection at 60 mg/kg of HS9_DSB7_V19A.

FIGS. 40A-C provides the results from oral glucose tolerance tests (day 0 (A), day 2 (B), day 7 (C), confirming the ability of a single, subcutaneous administration of HS9_DSB7_V19A.

FIG. 41 shows body-weight change over time in the oral glucose tolerance test of FIGS. 40A-C.

FIGS. 42A-C illustrates the results from oral glucose tolerance tests (day 0 (A), day 2 (B), day 7 (C).

FIG. 43 provides body-weight change over time in the oral glucose tolerance test of FIGS. 42A-C.

FIG. 44 shows body weight change in an intraperitoneal glucose tolerance test.

FIG. 45 demonstrates that weekly HS9_DSB7_V19A exhibited a dose dependent reduction in 4 hour fasting blood glucose compared to vehicle control in an intraperitoneal glucose tolerance test.

FIG. 46 shows that weekly dosed HS9_DSB7_V19A exhibited a dose dependent improvement in glucose tolerance as assessed by IPGTT at study day 22.

FIGS. 47A-B shows blood glucose levels in a diet-induced obesity mouse model.

FIGS. 48A-B shows body weight (grams) (A) and % change in body weight (B) in a diet-induced obesity mouse model.

FIG. 49 shows the change in body weight over time in a multiple dose study with HS9_DSB7_V19A.

FIGS. 50A-B show the effect of the GLP-1 component of HS9_DSB7_V19A on glycemic control in a weekly dosing setting, measuring fed glucose (A) and terminal fasting glucose (B).

DESCRIPTION OF THE SEQUENCES

Table 1 provides a listing of certain sequences referenced in present embodiments.

TABLE 1 SEQ Description Sequence ID NO GROUP A anti-PCSK9 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG EIHPSGGSTSYNQKFQG RVTMTRDT 1. antibody STSTVYMELSSLRSEDTAVYYCAR ERPLYASDL WGQGTTVTVSS heavy chain  (HS9_VH)  anti-PCSK9 DIQMTQSPSSLSASVGDRVTITC QASQDVKTAVA WYQQKPGKAPKLLIY SASYRYT GVPSRFSGSGSGTDFTFT 2. antibody ISSLQPEDIATYYC QQRYSLWRT FGQGTKLEIK light chain  (HS9_Vk)  GLP-1                  *{circumflex over ( )}                   # 3. moiety with  HGEGTFTSDLSKQMEEECARLFIEWLKNGGPSSGAPPPGCG cysteine With the * designating a cysteine bridge to the linker, bridge and the {circumflex over ( )} designating a point mutation to reduce potency and match CDTP double (maximum efficacy without nausea side effect), and mutation the # designating a point mutation to optimize disulfide bonding and reduce aggregation Linker GGGGSGGGGSGGGGSA 4. pH QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG EIHPSGGSTSYNQKFQG RVTMTRDT 5. dependent STSTVYMELSSLRSEDTAVYYCAR ERPLYASDL WGQGTTVTVSS version of  anti-PCSK9  antibody  heavy chain  (PC9#2_FG_  VH)  pH DIQMTQSPSSLSASVGDRVTITC QASQDVHTAVA WYQQKPGKAPKLLIY HASYRYT GVPSRFSGSGSGTDFTFT 6. dependent ISSLQPEDIATYYC QQRYSLWRT FGQGTKLEIK version of  anti-PCSK9  antibody  light chain  (PC9#2_FG_  Vk)  GLP-1  * 7. moiety with HGEGTFTSDLSKQMEEECVRLFIEWLKNGGPSSGAPPPSCG cysteine  mutation to  create  disulfide  bridge to  linker  Antibody A QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG EIHPSGGSTNYNEKFKS RVTMTRDT 8. pH STSTVYMELSSLRSEDTAVYYCAR ERPLYASDL WGQGTTVTVSS dependent  heavy chain  (PC9_2_VH)  Antibody A DIQMTQSPSSLSASVGDRVTITC KASQDVHTAVA WYQQKPGKAPKLLIY HASYRYT GVPSRFSGSGSGTDFTFT 9. pH ISSLQPEDIATYYC QQRYSLWRT FGQGTKLEIK dependent  light chain  (PC9_2_Vk)  Antibody B QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG EIHPFGGRTNYNEKFKS RVTMTRDT 10. non pH STSTVYMELSSLRSEDTAVYYCAR ERPLYASDL WGQGTTVTVSS dependent  heavy chain  (PC9_1_VH)  Antibody B DIQMTQSPSSLSASVGDRVTITC RASQGISSALA WYQQKPGKAPKLLIY SASYRYT GVPSRFSGSGSGTDFTFT 11. non pH ISSLQPEDIATYYC QQRYSLWRT FGQGTKLEIK dependent  light chain  (PC9_1_Vk)  GLP-1 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS 12. moiety  (exenatide  (Exe4))  GLP-1 HGEGTFTSDLSKQMEEECVRLFIEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSA 13. moiety  DSB#7 with  linker  (PC9_HS9_  DSB#7)  anti-PCSK9 SYYMH 14. antibody  heavy chain  CDR1  (PC9_2_HS9)  anti-PCSK9 EISPSGGSTSYNQKFQG 15. antibody  heavy chain  CDR2  (PC9_2_HS9_  VH)  anti-PCSK9 EIHPSGGSTSYNQKFQG 16. antibody  heavy chain  CDR2  (PC9#2_FG_  VH)  anti-PCSK9 EIHPSGGRTNYNEKFKS 17. antibody  heavy chain  CDR2  (PC9_2_VH)  anti-PCSK9 EISPFGGRTNYNEKFKS 18. antibody  heavy chain  CDR2  (PC9_1_VH)  anti-PCSK9 ERPLYASDL 19. antibody  heavy chain  CDR3  anti-PCSK9 QASQDVKTAVA 20. antibody  light chain  CDR1  (PC9_2_HS9_  Vk)  anti-PCSK9 QASQDVHTAVA 21. antibody  light chain  CDR1  (PC9#2_FG_  Vk)  anti-PCSK9 KASQDVKTAVA 22. antibody  light chain  CDR1  (PC9_2_Vk)  anti-PCSK9 RASQGISSAL A 23. antibody  light chain  CDR1  (PC9_1_Vk)  anti-PCSK9 SASYRYT 24. antibody  light chain  CDR2  (PC9_2_HS_Vk)  (PC9_1_Vk)  anti-PCSK9 HASYRYT 25. antibody  light chain  CDR2  (PC9#2_FG_Vk)  (PC9_2_Vk)  anti-PCSK9 QQRYSLWRT 26. antibody  light chain  CDR3  linker repeat GGGGS 27. GLP-1 HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG 28. moiety from  dulaglitude  (GLP-1 L)  Human GLP-1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 29. (7-37)  GLP-1 with HGEGTFTSCLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGGGGGGGCGG 30. disulfide  (DSB) #1  DSB#1  GLP-1 with HGECTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGGGGGGGGCG 31. DSB#2  GLP-1 with HGEGTFTSDLSKQMEEECVRLFIEWLKNGGPSSGAPPGC 32. DSB#3  GLP-1 with HGEGTFTSDLSKQMEEECVRCFIEWLKNGGPSSGAGGCS 33. DSB#4  GLP-1 with HGEGTFTSCLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGGGGGCGG 34. DSB#5  GLP-1 with HGEGTFTSCLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGGGGGGCGGG 35. DSB#6  GLP-1 with HGEGTFTSDLSKQMEEECVRLFIEWLKNGGPSSGAPPPGCG 36. DSB#7  GLP-1 with HGEGTFTSDLSKQMEEECVRLFIEWLKNGGPSSGAPPGGCG 37. DSB#8  GLP-1 with HGEGTFTSDLSKQMEEEAVRCFIEWLKNGGPSSGAPPCGG 38. DSB#9  GLP-1 with HGEGTFTSDLSKQMEEEAVRCFIEWLKNGGPSSGAPPGCG 39. DSB#10  GLP-1 with HGEGTFTSDLSKQMEEEAVRLFIECLKNGGPSSGACGGS 40. DSB#11  GLP-1 with HGEGTFTSDLSKQMEEEAVRLFIECLKNGGPSSGAPCPS 41. DSB#12  GLP-1 with HGEGTFTSDLSKQMEEEAVRLFIECLKNGGPSSGAPPCS 42. DSB#13  PC9#2_GLP1 HGEGTFTDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVTITCKASQ 43. VL with the DVHTAVAWYQQKPGKAPLLIYHASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRTFGQG linker of TKLEIKR SEQ ID NO: 4  underlined  (also  referenced  as  PC9_2_GLP1)  DSB#7 H V EGTFTSDLSKQMEEECVRLFIEWLKNGGPSSGAPPPGCG 44. Variant G2V  DSB#7 HGEGTFTSDLSKQM A EECVRLFIEWLKNGGPSSGAPPPGCG 45. Variant  E15A  DSB#7 HGEGTFTSDLSKQMEEECVRLFIEW I KNGGPSSGAPPPGCG 46. Variant  L261  HS9_DSB7_

GGGGSGGGGSGGGGSADIQMTQSPSSL 47. V19A VL SASVGDRVTITC QASQDVKTAVA WYQQKPGKAPKLLIY SASYRYT GVPSRFSGSGSGTDFTFTISSLQPEDIATY YC QQR YSLWRT FGQGTKLEIK PC9_2_DSB#1

GGGGSGGGGSGGGGSADIQMT 48. VL QSPSSLSASVGDRTITC KASQDVHTAVA WYQQKPGKAPKLLIY HASYRYT GVPSRFSGSGSGTDFTFTISSLQ PEDIATYYC QQRYSLWRT FGQGTKLEIKR PC9_2_DSB#3

GGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRV 49. VL TITC KASQDVHTAVA WYQQKPGKAPKLLIY HASYRYT GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQRYS LWRT FGQGTKLEIKR PC9_2_NGS#7

GGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVTITC KASQ 50. VL DVHTAVA WYQQKPGKAPKLLIY HASYRYT GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQRYSLWRT FGQG TKLEIKR PC9_2_DSB#7

GGGGSGGGGSGGGGSADIQMTQSP 51. VL SSLSASVGDRVTITC KASQDVHTAVA WYQQKPGKAPKLLIY HASYRYT GVPSRFSGSGSGTDFTFTISSLQPEDIAT YYC QQR YSLWRT FGQGTKLEIKR HS9_DSB#7

GGGGSGGGGSGGGGSADIQMT 52. VL QSPSSLSASVGDRVTITC QASQDVKTAVA WYQQKPGKAPKLLIY SASYRYT GVPSRFSGSGSGTDFTFTISSLQPEDI ATYYC QQR YSLWRT FGQGTKLEIKR GROUP B VH AB1 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG EIHPSGGSTNYNEKFKS RVTMTRDT 53. (pH-Dep) STSTVYMELSSLRSEDTAVYYCAR ERPLYASDL WGQGTTVTVSS VL AB1 DIQMTQSPSSLSASVGDRVTITC KASQDVHTAVA WYQQKPGKAPKLLIY HASYRYT GVPSRFSGSGSGTDFTFT 54. (pH-Dep) ISSLQPEDIATYYC QQRYSLWRT FGQGTKLEIK VH AB2 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMHWVRQAPGQGLEWMGEISPFGGRTNYNEKFKSRVTMTRDT 55. L1L3 STSTVYMELSSLRSEDTAVYYCAR ERPLYASDL WGQGTTVTVSS VL AB2 DIQMTQSPSSLSASVGDRVTITC RASQGISSALA WYQQKPGKAPKLLIY SASYRYT GVPSRFSGSGSGTDFTFT 56. L1L3 ISSLQPEDIATYYC QQRYSLWRT FGQGTKLEIK VH AB3 EVQLQQSGPELVKPGASVKISCKASGYTFT DYYMN WVKQSHGKSLEWIGDINPNNGGTTYNQKFKGKATLTVDK 57. 4A5 STSTVYMELSSLRSEDTAVYYCAR WLLFAY WGQGTTVTVSA VL AB3 DIVMTQSQKFMSTSVGDRVSVTC KASQNVFTNVA WYQQKPGQSPKALIY SASYRY SGVPDRFTGSGSGTDFTLT 58. 4A5 ISNVLSEDLAEYFC QQFYSYPYT FGGGTKLEIR VH AB4 QVQLQQPGAELVKPGASVKLSCKASGYTFT SYWMHWVKQRPGQGLEWIGEINPSNGRTNYNEKFKSKATLTVDK 59. 5A10 SSSTAYMQLSSLTSEDSAVYYCARER PLYAMDY WGQGTSVTVSS VL AB4 DIVMTQSHKFMSTSVGDRVSITC KASQDVSTAVA WYQQKPGQSPKLLIY SASYRY TGVPDRFTGSGSGTDFTFT 60. 5A10 ISSVQAEDLAVYYC QQRYSTPRT FGGGTKLEIKR VH AB4 EVQLQQSGPELVKPGASVKISCKASGYTFT DYYMNWVKQSHGKSLEWIGDINPNNGGTSYNQKFKGKATLTVDK 61. 6F6 SSSTAYMELRSLTSEDSAVYYCAGG GIYYRYDRNYFDY WGQGTTLTVSS VL AB4 DIQMTQTTSSLSASLGDRVTISC SASQGISNYLN WYQQKPDGTVKLLIY YTSSLHS GVPSRFSGSGSGTDYSLT 62. 6F6 ISNLEPEDIATYYC QQYSKLPFT FGSGTKLEIK VH AB4 EVKLVESEGGLVQPGSSMKLSCTASGFTFS DYYMA WVRQVPEKGLEWVA NINYDGSNTSYLDSLKSRFIISRDN 63. 7D4 AKNILYLQMSSLKSEDTATYYCAREKFA AMDY WGQGTSVTVSS VL AB4 DIVMTQSHKFMSTSFGDRVSITC KASQDVSNALA WYQQKPGHSPKLLIF SASYRYT GVPDRFTGSGSGTDFTFT 64. 7D4 ISSVQAEDLAVYYC QQHYSTPWT FGGGTKLEIKR GROUP C VH QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYGMH WVRQAPGKGLKWVA VIWYDGSDKYYADSVKG RFTISHDN 65. A74 30A4 SKNTLYLQMNSLRAEDTAVYYCAR ETGLPKLYYYGMDV WGQGTTVTVSS VH A85 QVQLQESGPGLVKPSQTLSLTCTVS GGSISSSDYYWS WIRQHPGKGLEWIG YIYYSGSTYYNPSLKS RITISVD 66. 3C4 TSKNLFSLKLSSVTAADTAVYYCAR GGVTTYYYAMDV WGQGTTVTVSS VH EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYAMN WVRQAPGKGLEWVS TISGSGDNTYYADSVKG RFTISRDN 67. A71 23B5 SKNTLYLQMNSLRAEDTAVYYCAK KFVLMVYAMLDY WGQGTLVTVSS VH EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYAMN WVRQAPGKGLEWVG TISGSGGNTYYADSVKG RFTISRDN 68. A72 25G4 SKNTLYLQMNSLRAEDTAVYYCAK KFVLMVYAMLDY WGQGTLVTVSS VH EVQLVESGGGLVKPGGSLRLSCAAS GFTFSSYSMN WVRQAPGKGLEWVS SISSSSSYISYADSVKG RFTISRD 69. A67 31H4 NAKNSLYLQMNSLRAEDTAVYFCAR DYDFWSAYYDAFDV WGQGTMVTVSS VH QVQLQESGPGLVKPSQTLSLTCTVS GGSISSGGYYWS WIRQHPGKGLEWIG YIYNSGSTYYNPSLKS RVTISVD 70. A87 27B2 TSKNQFSLKLSSVTAADTAVYYCAR EDTAMVPYFDY WGQGTLVTVSS VH QVQLVQSGAEVKKPGASVKVSCKAS GYTFPSYGIS WVRQAPGQGLEWMG WISAYNGNTNYAEKLQG RVTMTTDT 71. A58 25A7 STSTAYMEVRSLRSDDTAVFYCAR GYVMDV WGQGTTVTVSS VH A52 27H5 QVQLVQSGAEVKRPGASVKVSCKAS GYTLTSYGIS WVRQAPGQGLEWMG WISVYNGNTNYAQKVQG RVTMTTDT  72. STSTVYMELRSLSSDDTAVYYCAR GYGMDV WGQGTTVTVSS VH A51 26H5 QVQLVQSGAEVKKPGASVKVSCKAS GYTLTSYGIS WVRQAPGQGLEWMG WISFYNGNTNYAQKVQG RVTMTTDT  73. STSTVYMELRSLRSDDTAVYYCAR GYGMDV WGQGTTVTVSS VH A53 31D1 QIQLVQSGAEVKKPGASVKVSCKAS GYTLTSYGIS WVRQAPGQGLEWMG WISFYNGNTNYAQKVQG RVTMTTDT  74. STSTVYMELRSLRSDDTAVYFCAR GYGMDV WGQGTTVTVSS VH A48 20D10 QIQLVQSGAEVKKPGASVKVSCKAS GYPLTSYGIS WVRQAPGQGLEWMG WISAYNGNTNYAQKVQG SVTMTTDT  75. STSTVYMELRSLRSDDTAVYYCAR GYGMDV WGQGTTVTVSS VH A54 27E7 QVQLVQSGAEVKKPGASLKVSCKAS GYSLTSYGIS WVRQAPGQGLEWMG WISAYNGNTNYAQKVQG RVTMTTDT  76. STSTVYMEVRSLRSDDTAVYYCAR GYGMDV WGQGTTVTVSS VH A55 30B9 QVQLVQSGAEVKKPGASVKVSCKAS GYPLTSYGIS WVRQAPGQGLEWMG WISAYNGNTNYAQKVQG RVTMTTD  77 STSTVYMELRSLRSDDTAVYYCAR GYGMDV WGQGTTVTVSS VH A56 19H9 QVQLVQSGAEVKKPGASVKVSCKAS GYALTSYGIS WVRQAPGQGLEWMG WISAYNGNTNYAQKVQG RVTMTTDT  78. STSTVYMELRSLRSDDTAVYYCAR GYGMDV WGQGTTVTVSS VH A49 21B12 QVQLVQSGAEVKKPGASVKVSCKAS GYTLTSYGIS WVRQAPGQGLEWMG WVSFYNGNTNYAQKLQG RGTMTTDP  79. STSTAYMELRSLRSDDTAVYYCAR GYGMDV WGQGTTVTVSS VH A57 17C2 QVQLVQSGAEVKKPGASVKVSCKAS GYSFTSYGIS WVRQAPGQGLEWMG WVSAYNGMTNYAQKFQG RVTMTTDT  80. STSTAYMELRSLRSDDTAVYYCAR GYVMDV WGQGTTVTVSS VH A50 23G1 QVQLVQSGAEVKKPGASVKVSCKAS GYTLTSYGIS WVRQAPGQGLEWMG WVSFYNGNTNYAQKLQG RGTXTTDP 81. STSTAYMELRSLRSDDTAVYYCAR GYGMDV WGQGTTVTVSS Vh A91 13H1 QVQLQQSGPGLVKPSQTLSLTCAIS GDSVSSNSAAMN WIRQSPSRGLEWLG RTYYRSKWYKNYSVSVKS RITIN  82. PDTSKNQFSLQLNSVTPGDTAVYYCAR GGPTAAFDY KGQGTLVTVSS VH A64 9C9 EVQLVESGGGLVQPGGSLRLSCVVS GFTFSSYWMS WVRQAPGKGLEWVA NIKQDGSEKYYVDSVKG RFTISRDN  83. AKNSLYLQMNSLRAEDTAVYYCAR ESNWGFAFDI WGQGTMVTVSS VH A62 9H6 EVQLVESGGGLVQPGGSLRLSCAAS GFTFSRYWMS WVRQAPGKGLEWVA NIKHDGSEKYYVDSVKG RFTISRDN  84. AKNSLYLQMNSLRAEDTAVYYCAR ESNWGFAFDV WGHGTMVTVSS VH A89 31A4 QVQLQQWGAGLLKPSETLSLTCAVY GGSFSAYYWN WIRQPPGKGLEWIG EINHSGRTDYNPSLKS RVTISVDTS  85. KKQFSLKLNSVTAADTAVYYCARG QLVPFDY WGQGTLVTVSS VH A65 1A12 EVQLVESGGGLVQPGGSLRLSCAAS GLTFSNFWMS WVRQAPGKGLEWVA NIKQDGSEKYYVDSVKG RFTISRDN  86. AKNSLYLQMNSLRAEDTAVYSCTR ESNWGFAFDI WGQGTMVTVSS VH A79 16F12 QVHLVESGGGVVQPGRSLRLSCAAS GFTFNSFGMH WVRQAPGKGLEWVA LIWSDGSDEYYADSVKG RFTISRDN  87. SKNTLYLQMNSLRAEDTAVYYCAR AIAALYYYYGMDV WGQGTTVTVSS VH A80 22E2 QVQLVESCCGVVQPCRSLRLSCAAS GFTFSSFGMH WVRQAPGKCLEWVA LIWNDGSNKYYADSVKG RFTISRDN  88. SKNTLYLQMNSLRAEDTAVYYCAR AIAALYYYYGMDV WGQGTTVTVSS VH A76 27A6 QVHLVESGGGVVQPGRSLRLSCAAS GFTFNSFGMH WVRQAPGKGLEWVA LIWSDGSDKYYADSVKG RFTISRDN  89. SKNTLYLQMNSLRAEDTAVYYCAR AIAALYYYYGMDV WGQGTTVTVSS VH A77 28B12 QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSFGMH WVRQAPGKGLEWVA LIWNDGSNKYYADSVKG RFTISRDN  90. SKNTLYLQMNSLRAEDTAVYYCAR AIAALYYYYGMDV WGHGTTVTVSS VH A78 28D6 QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSFGM HWVRQAPGKGLEWVA LIWNDGSNKYYADSVKG RFTISRDN  91. SKNTLYLQMNSLRAEDTAVYYCAR AIAALYYYYGMDV WGQGTTVTVSS VH A83 31G11 QVQLVESGGGVVQPGRSLRLSCAAS GFTFRSYGM HKVRQAPGKGLEWVA LIWHDGSNTYYVDSVKG RFTISRDN  92. SKNTLYLQMNSLRAEDTAVYYCAR GIAVAYYYYGMDV WGQGTTVTVSS VH A69 13B5 EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYAMS WVRQAPGKGLEWVS TISGSGGRTYYADSVKG RFTISRDN  93. SKNTLYLQMNSLRAEDTAVYYCAK EVGSPFDY WGQGTLVTVSS VH A81 31B12 QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYGMH KWVQAPGKGLEWVAI IWYDGSNKYYADSVKG RFTISRDN  94. SKNTLYLQMNSLRAEDTAVYYCAR RGGLAARPGGMDV WGQGTTVTVSS VH A60 3B6 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYGIS KVRQAPGQGLEWMG WISTYNGNTNYAQKVQG RVTMTTDT  95. STSTAYMELRSLRSDDTAVYYCAR GYTRDY WGQGTLVTVSS VL 5 30A4 DIVMTQSPLSLSVTPGEPPSISC RSSQSLLHSNGYN FLNWYLQKPGQSPQLLIY LGSHRAS GVPDRFSGSGSGT  96. DFTLEISRVEAEDVGVYYC MQVLQTPFT FGPGTKVDIK VL 73C4 DIQMTQSPSSLSASVGDRVTITC RASQRISNYLS WYLQKPGIAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLT  97. ISSLQSEDFATYYC QQSYSTPLI FGGGTKVEIK VL 9 23B5 DILMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKVLIY AASSLQS GVPSRFSGSGSGTDFTLT  98. INSLQPEDFATYYC QQSYSSPIT FGQGTRLEIK VL 10 25G4 DIQMTQSPSSLSASVGDRVTITC RASQSISIYLN WYQQKPGKAPYLLIY AAASLQS GVPSRFSGSGSGTDFTLT  99. ISSLQPEDFATYYC QQSYSAPIT FGQGTRLEIK VL 12 31H4 QSVLTQPPSVSGAPGQRVTISC TGSSSNIGAGYDVH WYQQLPGTAPKLLIS GNSNRPS GVPDRFSGSKSGTSAS  100. LAITGLQAEDEADYYC QSYDSSLSGSV FGGGTKLTVL VL 13 27B2 QSVLTQPPSVSGAPGQRVTISC TGSSSNIGAHYDVH WYQQVPGTAPKLLIY GNTYRPS GVPDRFSGSKSGTSAS  101. LAITGLQAEDEADYYC QSYDNSLSGVV FGGGTKLTVL VL 15 25A7 QSALTQPASVSGSPGQSITISC TGTSSD VGRYN SVS WYQHHPGKAPKVMIY EVSNRPS GVSTRFSGSKSGNTAS  102. LTISGLQAEDEADYYC SSYTSSSVV FGGGTKLTVL VL 16 27H5 QSALTQPASVSGSPGQSITISC TGTSSDVGGYNSVS WYQQHPGKPPKLMIY EVSNRPS GVSIRFSGSKSGNTAS  103. LTISGLQAEDEADYFC SSYTSTSMV FGGGTKLTVL VL 17 26H5 QSALTQPASVSGSPGQSITISC TGTSSDVGGYNSVS WYQQHPGKPPKLMIY EVSNRPS GVSIRFSGSKSGNTAS  104. LTISGLQAEDEADYFC SSYTSTSMV FGGGTKLTVL VL 18 31D1 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNSVSWYQQHPGKPPKLMIY EVSNRPS GVSNRFSGSKSGNTAS  105. LTISGLQAEDEADYFC SSYTSTSMVF GGGTKLTVL VL 19 20D10 QSALTQPASVSGSPGQSITISC TGTSSDVGGYNSVS WYQQYPGKPPKLKIY EVSNRPS GVSNRFSGSKSGNTAS  106. LTISGLQAEDEADYFC SSYTSTSMV FGGGTKLTVL VL 20 27K7 QSALTQPASVSGSPGQSITISC TGTSSDVGGYNSVS WYQQHPGKPPKLMIY EVSNRPS GVSNRFSGSKSGNTAS  107. LTISGLQAEDEADYFC SSYTSTSMV FGGGTKLTVL VL 21 30B9 QSALTQPASVSGSPGQSITISC TGTSSDVGGYNSVS WYQQHPGKPPKLMIY EVSNRPS GVSNRFSGSKSGNTAS  108. LTISGLQAEDEADYFC SSYTSTSMV FGGGTKLTVL VL 22 19H9 QSALTQPASVSGSPGQSITISC TGTNSDVGGYNSVS WYQQHPGKPPKLMIY EVSNRPS GISNRFSGSKSGNTAS  109. LTISGLQAEDEADYFC SSYTSTSMV FGGGTKLTVL VL 23 21B12 QSALTQPASVSGSPGQSITISC TGTSSDVGGYNSVS WYQQHPGKAPKLMIY EVSNRPS GVSXRFSGSKSGNTAS  110. LTISGLQAEDEADYYC NSYTSTSMV FGGGTKLTVL VL 24 17C2 QSALTQPASVSGSPGQSITISC TGTSSDVGAYNSVS WYQQHPGKAPKRMIY EVSNRPS GVSNRFSGSKSGNTAS  111. LTISGLQAEDEADYYC SSYTSTNMV FGGGTKLTVL VL 26 23G1 QSALTQPASVSGSPGQSITISC TGTSSDVGGYNSVS WYQQHPGKAPKRMIY EVTNRPS GVSNRFSGSKSGNTAS  112. LTISGLQAEDEADYYC NSYTSTNMV FGGGTKLTVL VL 28 13HT LSALTQPASVSGSPGQSITISC TGTSSDVGNYNLVS WYQQYSGKAPKLMIY EVSKRPS GVSNRFSGSKSGNTAS  113. LTISGLQAEDEADYYC CSYAGSSTLV FGGGTKLTVL VL 30 9C9 QSVLTQPPSASGTPGQRVTISC SGSSSNIGSKTVN WYQQVPGTAPKLLIY RNNQRPL GVPDRFSGSKSGTSASL  114. AISGLQSEDEADYYC AAWDDSLNWV FGGGTKLTVL VL 31 9H6 QSVLTQPPSASGPPGQRVTISC SGSSSNIGSNTVN WYQQLPGTAPKLLIY SNNRRPS GVPDRFSGSKSGTSASL  115. AISGLQSEDEADYYC AAWDDSLNWV FGGGTKLTVL VL 32 31A4 QSVLTQPPSASGTPGQRVTISC SGSSSNIGSNTVN WYQQLPGTAPKLLIY SNNQRPS GVPDRFSGSKSGTSASL  116. AISGLQSEDEADYYC AVWDDSLNGWV FGGGTKLTVL VL 33 1A12 QSVLTQPPSASGTPGQRVTISC SGSSSNIGSKTVN WYQQFPGTAPKLLIY SNNRRPS GVPDRFSGSKSGTSASL  117. AISGLQSEDEADYYC AAWDDSLNWV FGAGTKLTVL VL 35 16F12 QSVLTQPPSVSAAPGQKVTISC SGSSSNIGNNFVS WYQQLPGTAPKLLIY DYNKRPS GIPDRFSGSKSGTSATL  118. GITGLQTGDEADYYC GTWDSSLSAYV FGTGTRVTVL VL 36 22K2 QSVLTQPPSVSAAPGQKVTISC SGSSSNIGNNFVS WYQQLPGTAPKLLIY DYNKRPS GIPDRFSGSKSGTSATL  119. GITGLQTGDEADYYC GTWDSSLSGYV FGTGTRVTVL VL 37 27A6 QSVLTQPPSVSAAPGQKVTISC SGSSSNIGNNFVS WYQQFPGTAPKLLIY DYNKRPS GIPDRFSGSKSGTSATL  120. GITGLQTGDEADYYC GTWDSSLSSYV FGTGTRVTVL VL 38 28B12 QSVLTQPPSVSAAPGQKVTISC SGSSSNIGNNFVS WYQQLPGTAPKLLIY DYNKRPS GIPDRFSGSKSGTSATL 121. GITGLQTGDEADYYC GTWDSSLSSYV FGTGTRVTVL VL 39 28D6 QSVLTQPPTVSAAPCQKVTISC SGSSSNIGNNFVS WYQQLPCTAPKLLIY DYNKRPS CIPDRFSCSKSCTSATL  122. GITGLQTGDEADYYC GTWDSSLSGYV FGTGTRVTVL VL 40 31G11 QSVLTQPPSVSAAPGQKVTISC SGSSSNIGNNFVS WYQQLPGTAPKLLIY DSNKRPS GIPDRFSGSKSGTSATL  123. DITGLQTGDEADYYC GTWDSSLSAYV FGTGTKVTVL VL 42 13B5 QSVLTQPPSVSAAPGQKVTISC SGSNSNIGNNYVS WYQQLPGTAPKLLIY DNNKRPS GIPDRFSGSNSGTSATL  124. GITGLQTGDEADYYC GTWDSSLSAVV FGGGTKLTVL VL 44 31B12 SYELTQPPSVSVSPGQTARITC SGDKLGDKYAC WYQQKPGQSPVLVIY QNTKWPL GIPERFSGSKSGNTVTLTI  125. SGTQAMDEADYYC QAWDSSTVV FGGGTKLTVL VL 46 3B6 QPVLTQPLFASASLGASVTLTC TLSSGYSSYEVD WYQQRPGKGPRFVMR VDTGGIVGSKGE GIPDRFSVLGSGL  126. NRYLTIKNIQEEDESDYHC GADHGSGTNFVVV FGGGTKLTVL GROUP D  VH 66 QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYG MHWVRQAPGKGLEWVAF IGFDGSNI YYGDSVRGRIIISRDN 127. SENTLYLEMNSLRAEDTAVYYC AREKGLD WGQGTLVTVSS VH 90  EVQLVESGGGLVQPGGSLRLSCAAS GFTFNNYA MNWVRQAPGKGLDWVST ISGSGGTT XYADSVKGRFIISRDS  128. H1H316P SKHTLYLQMNSLRAEDTAVYYC AKDSNWGNFDL WGRGTLVTVSS VH 138 QVQLQESGPGLVKPSETLSLTCTVS GDSINTYY WSWFRQPPGKGLEWIGY IYYSGTT NYNPSLKSRVTISIDTP  129. RNQFSLKLISVTAADTAVTTC ARERITMIRGVTLYYYSYGMDV WGQGTTVTVSS VH 218  EMQLVESGGGLVQPGGSLRLSCAAS GFTFSSHW MKWVRQAPGKGLEWVAN INQDGSEK YYVDSVKGRFTISRDN  130. H1M300N AKNSLFLQMNSLRAEDTAVYYC ARDIVLMVYDMDYYYYGMDV WGQGTTVTVSS VH 2 QVQLVQSGGGLVQPGGSLRLSCAAS GFTLSSYD MHWVRQSTGKGLEWVSA IGSTGDT YYPGSVKGRFTITREKA  131. KNSVYLQMNSLRAGDTAVYYC VREGWEVPFDY WGQGTLVTVSS VH 18 EVQLVESGGGLVQPGGSLRLSCAAS GFTLSSYD MHWVRQSTGKGLEWVSA IGSTGDT YYPGSVKGRFTITREKA  132. KNSVYLQMNSLRAGDTAVYYC VREGWEVPFDY WGQGTLVTVSS VH 22 EVQLVESGGGLVQPGGSLRLSCAAS GFTLSSYD MHWVRQATGKGLEWVSA IGSTGDT YYPGSVKGRFTISRENA  133. KNSLYLQMNSLRAGDTAVYYC VREGWEVPFDY WGQGTLVTVSS VH 26 QVQLVQSGGGVVQPGRSLRLSCAAS GFTLSSYG MHWVRQAPGKGLEWVAF IGSDGSNI HYGDSVKGRIIISRDN  134. SENTLYLEMNSLRAEDTAMYYC AREKGLD WGQGTTVTVSS VH 42 QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYG MHWVRQAPGKGLEWVAF IGFDGSNI HYGDSVKGRIIISRDN  135. SENTLYLEMNSLRAEDTAMYYC AREKGLD WGQGTLVTVSS VH 46 QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYG MHWVRQAPGKGLEWVAV IGFDGSNI YYADSVKGRFTISRDN  136. SKNTLYLQMNSLRAEDTAVYYC AREKGLD WGQGTLVTVSS VH 50 QVQLQESGGGVVQPGRSLRLSCAAS GFTFSSYG MHWVRQAPGKGLEWVAF IGFDGSNI YYGDSVRGRIIISRDN  137. SENTLYLEMNSLRAEDTAVYYC AREKGLD KGQGTLVTVSS VH 70 QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYG MHWVRQAPGKGLEWVAV IGFDGSNI YYADSVKGRFTISRDN  138. SKNTLYLQMNSLRAEDTAVYYC AREKGLD WGQGTLVTVSS VH 94 EVQLVESGGGLVQPGGSLRLSCAAS GFTFNNYA MSWVRQAPGKGLEWVSA ISGSGGTT YYADSVKGRFTISRDN  139. SKNTLYLQMNSLRAEDTAVYYC AKDSNWGNFDL WGRGTLVTVSS VH 98 QVQLVQSGGGLVQPGGSLRLSCAVS GFTLSSYD MHWVRQPTGKGLEWVSA IGSTGDT YYPGSVKGRFTISRENA  140. KNSLYLQMNSLRAGDTAVYYC AREGWDVPFDF WGQGTLVTVSS VH 114 EVQLVESGGGLVQPGGSLRLSCAVS GFTLSSYD MHWVRQPTGKGLEWVSA IGSTGDT YYPGSVKGRFTISRENA  141. KNSLYLQMNSLRAGDTAVYYC AREGWDVPFDF WGQGTLVTVSS VH 118 EVQLVESGGGLVQPGGSLRLSCAAS GFTLSSYD MHWVRQATGKGLEWVSA IGSTGDT YYPGSVKGRFTISRENA  142. KNSLYLQMNSLRAGDTAVYYC AREGWDVPFDF WGQGTLVTVSS VH 122 QVQLQESGPGLVKPSETLSLTCTVS GDSINTYY WSWIRQPPGKGLEWIGY IYYSGTT NYNPSLKSRVTISVDTS  143. KNQFSLKLSSVTAADTAVYYC ARERITMIRGVTLYYYSYGMDV WGQGTTVTVSS VH 142 QVQLQESGPGLVKPSETLSLTCTVS GDSINTYY WSWIRQPPGKGLEWIGY IYYSGTT NYNPSLKSRVTISVDTS  144. KNQFSLKLSSVTAADTAVYYC ARERITMIRGVTLYYYSYGMDV WGQGTTVTVSS VH 146 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTNYG TSWVRQAPGQGLELMGW ISGYNGNT NYAQELQARVTMTTDT  145. STSTAYMELRNLRSDDTAVYYC ARDRVVVAAANYYFYSMDV WGQGTTVTVSS VH 162 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTNYG TSWVRQAPGQGLELMGW ISGYNGNT NYAQELQARVTMTTDT 146. STSTAYMELRNLRSDDTAVYYC ARDRVVVAAANYYFYSMDV WGQGTTVTVSS VH 166 QVQLVQSGAEVKKPGASVKVSCKAH GYTFTNYG ISWVRQAPGQGLEWMGW ISGYNGNT NYAQKLQGRVTMTTDT  147. STSTAYMELRSLRSDDTAVYYC ARDRVVVAAANYYFYSMDV WGQGTTVTVSS VH 170 QVHLKESGPTLVKPTQTLTLTCTFS GFSLITSGVG VGWIRQPPGKALEWLAL IYWMGDK RYSPSLKSRLTITKD  148. TSKNQVVLTMTNMDPVDTATYYC AHRITETSYYFYYGMDV WGQGTTVTVSS VH 186 QITLKESGPTLVKPTQTLTLTCTFS GFSLITSGVG VGWIRQPPGKALEWLAL IYWNGDK RYSPSLKSRLTITKD  149.  TSKNQVVLTMTNMDPVDTATYYC AHRITETSYYFYYGMDV WGQGTTVTVSS VH 190 QITLKESGPTLVKPTQTLTLTCTFS GFSLITSGVG VGWIRQPPGKALEWLAL IYWNGDK RYSPSLKSRLTITKD 150. TSKNQVVLTMTNMDPVDTATYYC AHRITETSYYFYYGMDV WGQGTTVTVSS VH 194 QITLKESGPTLVKPTQTLTLTCTFS GFSLSTSGVG VGWIRQPPGKALEWLAL IYWNSDK RYSPSLKSRLTITKD 151. TSKNQVVLTMTNMDPVDTATYYC AHRHDSSSYYFYYGMDV WGQGITVTVSS VH 210 QITLKESGPTLVKPSQTLTLTCTFS GFSLSTSGVG VGWIRQPPGKALEWLAL IYWNSDK RYSPSLKSRLTITKD  152. TSKNQVVLTMTNMDPVDTATYYC AHRHDSSSYYFYYGMDV WGQGTTVTVSS VH 214 QITLKESGPTLVKPTQTLTLTCTFS GFSLSTSGVGV GWIRQPPGKALEWLAL IYWNSDK RYSPSLKSRLTITKD  153. TSKNQVVLTMTNMDPVDTATYYC AHRHDSSSYYFYYGMDV WGQGTTVTVSS VH 234 EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSHW MKWVRQAPGKGLEWVAN INQDGSEK YYVDSVKGRFTISRDN 154. AKNSLFLQMNSLRAEDTAVYYC ARDIVLMVYDMDYYYYGMDV WGQGTTVTVSS VH 238 EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSHW MSWVRQAPGKGLEWVAN INQDGSEK YYVDSVKGRFTISRDN  155. AKNSLYLQMNSLRAEDTAVYYC ARDIVLMVYDMDYYYYGMDV WGQGTTVTVSS VH 242 QVQLVESGGGVVQPGRSLRLSCAVS GFTFSSYG MHWVRQAPGKGLEWVAA ISYDGSNK YYVDSVKGRFTISRDN  156. SKKTLYLQMNSLRAEDTAVYNC AKNIVLVMYDIDYHYYGMDV WGQGTTVTVSS VH 258 QVQLVESGGGVVQPGRSLRLSCAVS GFTFSSYG MHWVRQAPGKGLEWVAA ISYDGSNK YYVDSVKGRFTISRDN  157. SKKTLYLQMNSLRAEDTAVYNC AKNIVLVMYDIDYHYYGMDV WGQGTTVTVSS VH 262 QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYG MHWVRQAPGKGLEWVAV ISYDGSNK YYADSVKGRFTISRDN  158. SKKTLYLQMNSLRAEDTAVYYC AKNIVLVMYDIDYHYYGMDV WGQGTTVTVSS VH 266 QVQLVESGGGVVQPGRSLRLSCAVS GFTFSSYG MHWVRQAPGKGLEWVAA ISYDGSNK YYVDSVKGRFTISRDN  159. SKKTLYLQMNSLRAEDTAVYNC AKNIVLVMYDIDYHYYGMDV WGQGTTVTVSS VH 282 QVQLVESGGGVVQPGRSLRLSCAVS GFTFSSYG MHWVRQAPGKGLEWVAA ISYDGSNK YYVDSVKGRFTISRDN  160. SKKTLYLQMNSLRAEDTAVYNC AKNIVLVMYDIDYHYYGMDV WGQGTTVTVSS VH 286 QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYG MHWVRQAPGKGLEWVAV ISYDGSNK YYADSVKGRFTISRDN 161. SKNTLYLQMNSLRAEDTAVYYC AKNIVLVMYDIDYHYYGMDV WGQGTTVTVSS VH 290 QITLKESGPTLVKPTQTLTLTCTFS GFSLSASG VGVGWFRQPPGKALEWLAL IYWNDDK RYSPSLKNSLTITKD 162. TSKNQVVLTMTNMDPVDTATYYC AHRIHLWSYFYYGMDV WGQGTTVTVSS VH 306 QITLKESGPTLVKPTQTLTLTCTFS GFSLSASG VGVGWFRQPPGKALEWLAL IYWNDDK RYSPSLKNSLTITKD 163. TSKNQVVLTMTNMDPVDTATYYC AHRIHLWSYFYYGMDV WGQGTTVTVSS VH 310 QITLKESGPTLVKPTQTLTLTCTFS GFSLSASG VGVGWIRQPPGKALEWLAL IYWNDDK RYSPRLKNSLTITKD 164. TSKNQVVLTMTNMDPVDTATYYC AHRIHLWSYFYYGMDV WGQGTTVTVSS VH 314 QVQLVQSGPEVKNPGASVKVSCKAS GYTFTTYG ISWVRQAPGQGLEWMGW ISGYHGKT NDAQKFQDRVAMTTDT  165. STSTAYMELRSLRSDDTAIYYC SRDRLVVPPALNYSYYVMDV WGQGTTVTVSS VH 330 QVQLVQSGPEVKMPGASVKVSCKAS GYTFTTYG ISWVRQAPGQGLEWMGW ISGYHGKT NDAQKFQDRVAMTTDT  166. STSTAYMELRSLRSDDTAIYYC SRDRLVVPPALNYSYYVMDV WGQGTTVTVSS VH 334 QVQLVQSGPEVKMPGASVKVSCKAS GYTFTTYG ISWVRQAPGQGLEWMGW ISGYHGKT NYAQKFQGRVTMTTDT  167. STSTAYMELRSLRSDDTAVYYC SRDRLVVPPALNYSYYVMDV WGQGTTVTVSS VH 338 EVQLVESGGGLVKPGGSLRLSCAAS GFTFSSYS MDWVRQAPGKGLEWVSS ISSSSSYI YYADSVKGRFTISRDT  168. AKNSLYLQMNSLRDEDTAVYYC AREGSSRLFDY WGQGTLVTVSS VH 354 EVQLVESGGGLVKPGGSLRLSCAAS GFTFSSYS MDWVRQAPGKGLEWVSS ISSSSSYI YYADSVKGRFTISRDT  169. AKNSLYLQMNSLRDEDTAVYYC AREGSSRLFDY WGQGTLVTVSS VH 358 EVQLVESGGGLVKPGGSLRLSCAAS GFTFSSYS MNWVRQAPGKGLEWVSS ISSSSSYI YYADSVKGRFTISRDN  170. AKNSLYLQMNSLRAEDTAVYYC AREGSSRLFDY WGQGTLVTVSS VH 362 QVHLVESGGGLVKPGGSLRLSCAAS GFTFSDHY MSWIRQAPGKGLEWISY ISNDGGTK YYVDSVEGRFIISRDN  171. AKNSLYLHMNSLRADDTAVYYC ARDQGYIGYDSYYYYSYGMDV WGQGTTVTVAS VH 378 QVQLVESGGGLVKPGGSLRLSCAAS GFTFSDHY MSWIRQAPGKGLEWISY ISNDGGTK YYVDSVEGRFIISRDN  172. AKNSLYLHMNSLRADDTAVYYC ARDQGYIGYDSYYYYSYGMDV WGQGTTVTVSS VH 382 QVQLVESGGGLVKPGGSLRLSCAAS GFTFSDHY MSWIRQAPGKGLEWVSY ISNDGGTK YYADSVKGRFTISRDN  173. AKNSLYLQMNSLRAEDTAVYYC ARDQGYIGYDSYYYYSYGMDV WGQGTTVTVSS VH 386 EVQKVESGGGLVKPGGSLRLSCTAS GFTFSTYN MNWVRQAPGKGLEWVSS IRSSSNYI YYADSVKGRFTISRDN  174. AKNSLYLQMNSLRADDTAVYYC ARDGSSWYDYSDY WGQGTLVTVSS VH 402 EVQLVESGGGLVKPGGSLRLSCGAS GFTFSTYN MNWVRQAPGKGLEWVSS IRSSSNYI YYADSVKGRFTISRDN  175. AKNSLYLQMNSLRADDTAVYYC ARDGSSWYDYSDY WGQGTLVTVSS VH 406 EVQLVESGGGLVKPGGSLRLSCAAS GFTFSTYN MNWVRQAPGKGLEWVSS IRSSSNYI YYADSVKGRFTISRDN  176. AKNSLYLQMNSLRAEDTAVYYC ARDGSSWYDYSDY WGQGTLVTVSS VH 410 EVQLVESGGGLVKPGGSLRLSCTAE GFTFSTYN MNWVRQAPGKGLEWVSS IRSSSNYI YYADSVKGRFTISRDN  177. AKNSLYLQMNSLRADDTAVYYC ARDGSSWYDYSDY WGQGTLVTVSS VH 426 EVQLVESGGGLVKPGGSLRLSCTAS GFTFSTYN MNWVRQAPGKGLEWVSS IRSSSNYI YYADSVKGRFTISRDN  178. AKNSLYLQMNSLRADDTAVYYC ARDGSSWYDYSDY WGQGTLVTVSS VH 430 EVQLVESGGGLVKPGGSLRLSCAAS GFTFSTYN MNWVRQAPGKGLEWVSS IRSSSNYI YYADSVKGRFTISRDN  179. AKNSLYLQMNSLRAEDTAVYYC ARDGSSWYDYSDY WGQGTLVTVSS VH 434 EVQLVESGGGLVKPGGSLRLSCAAS GFTFSTYN MNWVRQAPGKGLEWVSS IRSSSNYI YYADSVKGRFTISRDN  180. AKSSLYLQMNSLRAEDTAVYYC ARDGSSWYDYSDY WGQGTLVTVSS VH 450 EVQLVESGGGLVKPGGSLRLSCAAS GFTFSTYN MNWVRQAPGKGLEWVSS IRSSSNYI YYADSVKGRFTISRDN  181. AKSSLYLQMNSLRAEDTAVYYC ARDGSSWYDYSDY WGQGTLVTVSS VH 454 EVQLVESGGGLVKPGGSLRLSCAAS GFTFSTYN MNWVRQAPGKGLEWVSS IRSSSNYI YYADSVKGRFTISRDN  182. AKNSLYLQMNSLRAEDTAVYYC ARDGSSWYDYSDY WGQGTLVTVSS VH 458 EVQLVESGGGLVKPGGSLRLSCAAS GFTFSTYN MNWVRQAPGKGLEWVSS IRSSSNYI YYADSVKGRFTISRDN  183. AKNSLYLQMNSLRADDTAVYYC ARDGSSWYDYSDY WGQGTLVTVSS VH 474 EVQLVESGGGLVKPGGSLRLSCTAE GFTFSTYN MNWVRQAPGKGLEWVSS IRSSSNYI YYADSVKGRFTISRDN 184. AKNSLYLQMNSLRADDTAVYYC ARDGSSWYDYSDY WGQGTLVTVSS VH 478 EVQLVESGGGLVKPGGSLRLSCAAS GFTFSTYN MNWVRQAPGKGLEWVSS IRSSSNYI YYADSVKGRFTISRDN  185. AKNSLYLQMNSLRAEDTAVYYC ARDGSSWYDYSDY WGQGTLVTVSS VH 482 EVQLVESGGGLVQPGGSLRLSCVVS GFTFGDYD MHWVRQATGRGLEWVSG IAPAGDT SYTGSVKGRFTISRENA  186. KNSLHLQMNSLTTGDTAIYYC AREDIAVPGFDY WGQGTLVTVSS VH 498 EVQLVESGGGLVQPGGSLRLSCVVS GFTFGDYD MHWVRQATGRGLEWVSG IAPAGDT SYTGSVKGRFTISRENA  187. KNSLHLQMNSLTTGDTAIYYC AREDIAVPGFDY WGQGTLVTVSS VH 502 EVQLVESGGGLVQPGGSLRLSCAAS GFTFGDYD MHWVRQATGKGLEWYSA IAPAGDT YYPGSVKGRFTISRENA  188. KNSLYLQMNSLRAGDTAVYYC AREDIAVPGFDY WGQGTLVTVSS VH 506 QILLVQSGPEVKEPGASVKVSCKAS GYTFTNYA ISWVRQVPGQGLEWMGW VSAYNGHT NYAHEVQGRVTMTTDT  189. STTTAYMELRSLRSDDTAMYYC ARGGVVVPVAPHFYNGMDV WGQGTTVTVSS VH 522 QVQLVQSGPEVKEPGASVKVSCKAS GYTFTNYA ISWVRQVPGQGLEWMGW VSAYNGHT NYAHEVQGRVTMTTDT  190. STTTAYMELRSLRSDDTAMYYC ARGGVVVPVAPHFYNGMDV WGQGTTVTVSS VH 526 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTNYA ISWVRQAPGQGLEWMGW VSAYNGHT NYAQKLQGRVTMTTDT  191. STSTAYMELRSLRSDDTAVYYC ARGGVVVPVAPHFYNGMDV WGQGTTVTVSS VH 530 EVQLVESGGGLVQPGGSLRLSCAAS GFTLSSYD MHWVRQATGKGLEWVSA IGSTGDT YYTGSVMGRFTISRDAA 192. KNSFYLEMNSLRVGDTAVYYC AREGIRTPYDY WGQGARVTVSS VH 546 EVQLVESGGGLVQPGGSLRLSCAAS GFTLSSYD MHWVRQATGKGLEWVSA IGSTGDT YYTGSVMGRFTISRDAA  193. KNSFYLEMNSLRVGDTAVYY CAREGIRTPYDY WGQGTLVTVSS VH 550 EVQLVESGGGLVQPGGSLRLSCAAS GFTLSSYD MHWVRQATGKGLEWVSA IGSTGDT YYPGSVKGRFTISRENA  194. KNSLYLQMNSLRAGDTAVYYC AREGIRTPYDY WGQGTLVTVSS VH 554 EVQLVESGGGLVQPGGSLRLSCAAS GFTLSSYD MHWVRQATGKGLEWVSA IGSTGDT YYTGSVMGRFTISRDAA  195. KNSFYLEMNSLRVGDTAVYYC AREGIRTPYDY WGQGARVTVSS VH 570 EVQLVESGGGLVQPGGSLRLSCAAS GFTLSSYD MHWVRQATGKGLEWVSA IGSTGDT YYTGSVMGRFTISRDAA  196. KNSFYLEMNSLRVGDTAVYYC AREGIRTPYDY WGQGTLVTVSS VH 574 EVQLVESGGGLVQPGGSLRLSCAAS GFTLSSYD MHWVRQATGKGLEWVSA IGSTGDT YYPGSVKGRFTISRENA  197. KNSLYLQMNSLRAGDTAVYYC AREGIRTPYDY WGQGTLVTVSS VH 578 EVQLVESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSG INWNSGSI GYADSVKGRFTISRDN  198. AKHSLYLQMNSLRPEDTALYYC VKEVTTGYYYGMDV WGQGTTVTVSS VH 594 EVQLVESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSG INWNSGSI GYADSVKGRFTISRDN  199. AKHSLYLQMNSLRPEDTALYYC VKEVTTGYYYGMDV WGQGTTVTVSS VH 598 EVQLVESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSG INWNSGSI GYADSVKGRFTISRDN  200. AKHSLYLQMNSLRPEDTALYYC VKEVTTGYYYGMDV WGQGTTVTVSS VH 602 EVQLLESGGGLVQPGGLSRLSCAAS GFTFSSYA MNWVRQAPGKGLDWVSG ISGNGGST YYADSVKGRFTISRDI 201. SKNTLYVQMHSLRVEDTAVYYC AKARYYDFWGGNFDL WGRGTQVTVSS VH 618 EVQLLESGGGLVQPGGLSRLSCAAS GFTFSSYA MNWVRQAPGKGLDWVSG ISGNGGST YYADSVKGRFTISRDI 202. SKNTLYVQMHSLRVEDTAVYYC AKARYYDFWGGNFDL WGRGTQVTVSS VH 622 EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVSA ISGNGGST YYADSVKGRFTISRDN  203. SKNTLYLQMNSLRAEDTAVYYC AKARYYDFWGGNFDL WGRGTLVTVSS VH 626 QVQLVQSGPEVKNPGASVKVSCKAS GYTFTTYG ISWVRQAPGQGLEWMGW ISGYNGKT NDAQKFQDRVAMTTDT  204. STSTAYMELRSLRSDDTAIYYC SRDRLVVPPALYYSYYVMDV WGQGTTVTVSS VH 642 QVQLVQSGPEVKNPGASVKVSCKAS GYTFTTYG ISWVRQAPGQGLEWMGW ISGYNGKT NDAQKFQDRVAMTTDT  205. STSTAYMELRSLRSDDTAIYYC SRDRLVVPPALYYSYYVMDV WGQGTTVTVSS VH 646 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTTYG ISWVRQAPGQGLEWMGW ISGYNGKT NDAQKLQDRVTMTTDT  206. STSTAYMELRSLRSDDTAVYYC SRDRLVVPPALYYSYYVMDV WGQGTTVTVSS VH 650 QVQLVQSGPEVKNPGASVKVSCKAS GYTFTTYG ISWVRQAPGQGLEWMGW ISGYNGKT NDAQKFQDRVAMTTDT  207. STSTAYMELRSLRSDDTAIYYC SRDRLVVPPALYYSYYVMDV WGQGTTVTVSS VH 666 QVQLVQSGPEVKNPGASVKVSCKAS GYTFTTYG ISWVRQAPGQGLEWMGW ISGYNGKT NDAQKFQDRVAMTTDT  208. STSTAYMELRSLRSDDTAIYYC SRDRLVVPPALYYSYYVMDV WGQGTTVTVSS   VH 670 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTTYG ISWVRQAPGQGLEWMGW ISGYNGKT NYAQKLQGRVTMTTDT 209. STSTAYMELRSLRSDDTAVYYC SRDRLVVPPALNYYYYVMDV WGQGTTVTVSS VH 674 QVQLVQSGPEVKNPGASVKVSCKAE GYTFTTYG ISWVRQAPGQGLEWMGW ISGYNGKT NDAQKFQDRVAMTTDT  210. STSTAYMELRSLRSDDTAIYYC SRDRLVVPPALYYYYYVMDV WGQGTTVTVSS VH 690 QVQLVQSGPEVKNPGASVKVSCKAS GYTFTTYG TSWVRQAPGQGLEWMGW ISGYNGKT NDAQKFQDRVAMTTDT  211. STSTAYMELRSLRSDDTAIYYC SRDRLVVPPALYYYYYVMDV WGQGTTVTVSS VH 694 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTTYG ISWVRQAPGQGLEWMGW ISGYNGKT NYAQKLQGRVTMTTDT  212. STSTAYMELRSLRSDDTAVYYC SRDRLVVPPALYYYYYVMDV WGQGTTVTVSS VH 698 QVHLVESGGGLVKPGGSLRLSCAAK GFTFSDHY MSWIRQAPGKGLEWISY ISNDGGTK YYVDSVEGRFIISRDN  213. AKNSLYLHMNSLRADDTAVYYC ARDQGYIGYDSYYYYSYGMDV WGQGTTVTVAS VH 714 QVQLVESGGGLVKPGGSLRLSCAAK GFTFSDHY MSWIRQAPGKGLEWISY ISNDGGTK YYVDSVEGRFIISRDN  214. AKNSLYLHMNSLRADDTAVYYC ARDQGYIGYDSYYYYSYGMDV WGQGTTVTVSS VH 718 QVQLVESGGGLVKPGGSLRLSCAAS GFTFSDHY MSWIRQAPGKGLEWVSY ISNDGGTK YYADSVKGRFTISRDN  215. AKNSLYLQMNSLRAEDTAVYYC ARDQGYIGYDSYYYYSYGMDV WGQGTTVTVSS VH 722 QILLVQSGPEVKEPGASVKVSCKAS GYTFTNYA ISWVRQVPGQGLEWMGW VSAYNGHT NYAHEVQGRVTMTTDT  216. STTTAYMELRSLRSDDTAMYYC ARGGVVVPVAPHFYNGMDV WGQGTTVTVSS VH 738 QVQLVQSGPEVKEPGASVKVSCKAS GYTFTNYA ISWVRQVPGQGLEWMGW VSAYNGHT NYAHEVOGRVTMTTDT  217. STTTAYMELRSLRSDDTAMYYC ARGGVVVPVAPHFYNGMDV WGQGTTVTVSS VH 742 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTNYA ISWVRQAPGQGLEWMGW VSAYNGHT NYAQKLQGRVTMTTDT  218. STSTAYMELRSLRSDDTAVYYC ARGGVVVPVAPHFYNGMDV WGQGTTVTVSS VL 68 DIVMTQSPDSLAVSLGERATINCKSS QSVFHTSNNKNY LVWYQQKPGQPPKLLLY MAS TRESGVPDRFSGSGSG  219. TDFTLTISSLQAEDVANYYC HQYYSIPWT FGQGTKVEIK VL 92  DIVMTQSPDSLAVSLGERATINCKSS QSVLYRSNNRNF LGWYQQKPGQPPNLLIY WAS TRESGVPDRFSGSGSG  220. H1H316P TDFTLTISSLQAEDVAVYYC QQYYTTPYT FGQGTKLEIK VL 140 DIQMTQSPSFLSASVGDRVTITCWAS QDISSY LAWYQQKPGIAPKLLIY AAS TLQSGVPSRFGGSGSGTEFTLT  221. ISSLQPEDFATYYC QQLNSYPRT FGQGTKVEIK VL 226  DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGNNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGT  222. H1M300N DFTLKISRVEAEDVGVYYC MQTLQTPLT FGGGTKVEIK VL 10 DIQMTQSPATLSVSPGERAALSCRAS QSVSSN LAWYHQKPGQAPRLLIY GAS TRATGIPARFSGIGSGTEFTLI  223. ISSLQSEDFAFYFC QQYNNWPPFT FGPGTKVEIKR VL 20 EIVMTQSPATLSVSPGERAALSCRAS QSVSSN LAWYHQKPGQAPRLLIY GAS TRATGIPARFSGIGSGTEFTLI 224. ISSLQSEDFAFYFC QQYNNWPPFT FGPGTKVDIK VL 24 EIVMTQSPATLSVSPGERATLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLT  225. ISSLQSEDFAVYYC QQYNNWPPFT FGPGTKVDIK VL 34 AIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQKPGKAPKLLIY KAS SLESGVPSRFSGSGSGTEFTLT  226. ISSLQPDDFATYYC QQYNSYYT FGQGTKVEIKR VL 44 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQKPGKAPKLLIY KAS SLESGVPSRFSGSGSGTEFTLT  227. ISSLQPDDFATYYC QQYNSYYT FGQGTKLEIK VL 48 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQKPGKAPKLLIY KAS SLESGVPSRFSGSGSGTEFTLT  228. ISSLQPDDFATYYC QQYNSYYT FGQGTKLEIK VL 58 AIQMTQSPDSLAVSLGERATINCKSS QSVFHTSNNKNY LVWYQQKPGQPPKLLLY WAS TRESGVPDRFSGSGSG  229. TDFTLTISSLQAEDVANYYC HQYYSIPWT FGQGTKVEIKR VL 72 DIVMTQSPDSLAVSLGERATINCKSS QSVFHTSNNKNY LAWYQQKPGQPPKLLIY WAS TRESGVPDRFSGSGSG  230. TDFTLTISSLQAEDVAVYYC HQYYSIPWT FGQGTKVEIK VL 82 DIQMTQSPDSLAVSLGERATINCKSS QSVLYRSNNRNF LGWYQQKPGQPPNLLIY WAS TRESGVPDRFSGSGSG  231. TDFTLTISSLQAEDVAVYYC QQYYTTPYT FGQGTKVEIKR VL 96 DIVMTQSPDSLAVSLGERATINCKSS QSVLYRSNNRNF LAWYQQKPGQPPNLLIY WAS TRESGVPDRFSGSGSG  232. TDFTLTISSLQAEDVAVYYC QQYYTTPYT FGQGTKVEIK VL 106 AIQLTQSPSSLSASVGDRVTITCRAS QDIRND LGWYQQKPGKAPKLLIY AAS SLQSGVPSRFSGSGSGTDFTLT  233. ISSLQPEDFATYYC LQDYNYPWT FGQGTKVEIKR VL 116 AIQMTQSPSSLSASVGDRVTITCRAS QDIRND LGWYQQKPGKAPKLLIY AAS SLQSGVPSRFSGSGSGTDFTLT  234. TSSLQPEDFATYYC LQDYNYPWT FGQGTKVEIK VL 120 AIQMTQSPSSLSASVGDRVTITCRAS QDIRND LGWYQQKPGKAPKLLIY AAS SLQSGVPSRFSGSGSGTDFTLT  235. ISSLQPEDFATYYC LQDYNYPWT FGQGTKVEIK VL 130 DIQMTQSPSFLSASVGDRVTITCWAS QDISSY LAWYQQKPGIAPKLLIY AAS TLQSGVPSRFGGSGSGTEFTLT  236. ISSLQPEDFATYYC QQLNSYPRT FGQGTKVEIKR VL 144 DIQMTQSPSSLSASVGDRVTITCRAS QDISSY LGWYQQKPGKAPKRLIY AAS SLQSGVPSRFSGSGSGTEFTLT  237. ISSLQPEDFATYYC QQLNSYPRT FGQGTKVEIK VL 154 AIQMTQSPLSLSVTLGQPASISCRSSQSLVYSDGDTYLNWFQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  238. AFTLKISGVEAEDVGVYYC MQATHWPRT FGQGTKVEIKR VL 164 DVVMTQSPLSLSVTLGQPASISCRSS QSLVYSDGDTY LNWFQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  239. AFTLKISGVEAEDVGVYYC MQATHWPRT FGQGTKVEIK VL 168 DVVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGDTY LNWFQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  240. DFTLKISRVEAEDVGVYYC MQATHWPRT FGQGTKVEIK VL 178 DIQMTQSPLSLPVTPGEPASISCRSS QSLLHSHGYDY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGT  241. DFTLKISRVEAEDVGVYYC MQALQTPLT FGGGTKVEIKR VL 188 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSHGYDY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGT  242. DFTLKISRVEAEDVGVYYC MQALQTPLT FGGGTKVEIK VL 192 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSHGYDY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGT  243. DFTLKISRVEAEDVGVYYC MQALQTPLT FGGGTKVKIK VL 202 DIQMTQSPLSLPVTPGEPASISCRSS QSLLHSHGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGGGSGT  244. DFTLKISRVEAEDVGIYYC MQALQTPLT FGGGTKVKIKR VL 212 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSHGYDY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGT  245. DFTLKISRVEAEDVGIYYC MQALQTPLT FGGGTKVKIK VL 216 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSHGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGT 246. DFTLKISRVEAEDVGVYYC MQALQTPLT FGGGTKVKIK VL 236 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGNNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGT  247. DFTLKISRVEAEDVGVYYC MQTLQTPLT FGGGTKVEIK VL 2-10 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGNNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGT  248. DFTLKISRVEAEDVGVYYC MQTLQTPLT FGGGTKVEIK VL 250 DIVMTOSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLOKPGOSPOLLIY LGF NRASGVPDRFSGSGSGT  249. DFTLKISRVEAEDVGVYYC MQALQTPLT FGGGTKVEIR VL 260 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGF NRASGVPDRFSGSGSGT  250. DFTLKISRVEAEDVGVYYC MQALQTPLT FGGGTKVEIK VL 264 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGF NRASGVPDRFSGSGSGT  251. DFTLKISRVEAEDVGVYYC MQALQTPLT FGGGTKVEIK VL 274 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGF NRASGVPDRFSGSGSGT  252. DFTLKISRVEAEDVGVYYC MQALQTPLT FGGGTKVEIR VL 284 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGF NRASGVPDRFSGSGSGT 253. DFTLKISRVEAEDVGVYYCM QALQTPLT FGGGTKVEIK VL 288 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNY LDWYLQKPGQSPQLLIY LGF NRASGVPDRFSGSGSGT  254. DFTLKISRVEAEDVGVYYC MQALQTPLT FGGGTKVEIK VL 298 DIVMTQSPLSLPVTPGEPASISCRSS QTLLHSNGYNY FDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGT  255. DFTLKISRVEAEDVGIYYC MQALQTPLT FGGGTKVEIR VL 308 DIVMTQSPLSLPVTPGEPASISCRSS QTLLHSHGYNY FDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGT  256. DFTLKISRVEAEDVGIYYC MQALQTPLTF GGGTKVEIK VL 312 DIVMTQSPLSLPVTPGEPASISCRSS QTLLHSNGYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGT 257. DFTLKISRVEAEDVGVYYC MQALQTPLT FGGGTKVEIK VL 322 DVVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGNTY LNWSQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  258. DFTLKISRVEAEDVGVYYC MQGTHWPYT FGQGTKLEIK VL 332 DVVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGNTY LNWSQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  259. DFTLKISRVEAEDVGVYYC MQGTHWPYT FGQGTKLEIK VL 336 DVVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGNTY LHWFQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  260. DFTLKISRVEAEDVGVYYC MQGTHWPYT FGQGTKLEIK VL 346 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQRPGKAPKLLIY KAS SLEGGVPSRFSGSGSGTEFTLT  261. ISSLQPEDFATYYC QQYNSYWYT FGQGTKLEIK VL 356 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQRPGKAPKLLIY KAS SLEGGVPSRFSGSGSGTEFTLT  262. ISSLQPEDFATYYC QQYNSYWYT FGQGTKLEIK VL 360 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQKPGKAPKLLIY KAS SLESGVPSRFSGSGSGTEFTLT  263. ISSLQPDDFATYYC QQYNSYWYT FGQGTKLEIK VL 370 KIVLTQSPGTLPLFPGERATLSCRAS QSVNNKF LAWYQQKSGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTL  264. TISGLEPEDFEVYYC QVYGNSLT LGGGTKVEIK VL 380 EIVLTQSPGTLPLFPGERATLSCRAS QSVNNKF LAWYQQKSGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTL  265. TISGLEPEDFEVYYC QVYGNSLT LGGGTKVEIK VL 384 EIVLTQSPGTLSLSPGERATLSCRAS QSVNNKF LAWYQQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTL 266. TISRLEPEDFAVYYC QVYGNSLT FGGGTKVEIK VL 394 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQIPGKAPKLLIY KAS SLENGVPSRFSGSGSGTEFTLI  267. ISSLQPDDFATYYC QQYISYSRT FGQGTKVEIK VL 404 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQIPGKAPKLLIY KAS SLENGVPSRFSGSGSGTEFTLI  268. ISSLQPDDFATYYC QQYISYSRT FGQGTKVEIK VL 408 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQKPGKAPKLLIY KAS SLESGVPSRFSGSGSGTEFTLT  269. ISSLQPDDFATYYC QQYISYSRT FGQGTKVEIK VL 418 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQIPGKAPKLLIY KAS SLENGVPSRFSGSGSGTEFTLI  270. ISSLQPDDFATYYC QQYISYSRT FGQGTKVEIK VL 428 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQIPGKAPKLLIY KAS SLENGVPSRFSGSGSGTEFTLI  271. ISSLQPDDFATYYC QQYISYSRT FGQGTKVEIK VL 432 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQKPGKAPKLLIY KAS SLESGVPSRFSGSGSGTEFTLT  272. ISSLQPDDFATYYC QQYISYSRT FGQGTKVEIK VL 442 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQVPGKAPKLLIY KAS SLENGVPSRFSGSGSGTEFTLI  273. ISSLQPDDFATYYC QQYISYSRT FGQGTKVEIK VL 452 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQVPGKAPKLLIY KAS SLENGVPSRFSGSGSGTEFTLI  274. ISSLQPDDFATYYC QQYISYSRT FGQGTKVEIK VL 456 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQKPGKAPKLLIY KAS SLESGVPSRFSGSGSGTEFTLT  275. ISSLQPDDFATYYC QQYISYSRT FGQGTKVEIK VL 466 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQIPGKAPKLLIY KAS SLENGVPSRFSGSGSGTEFTLI  276. ISSLQPDDFATYYC QQYISYSRT FGQGTKVEIK VL 476 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQIPGKAPKLLIY KAS SLENGVPSRFSGSGSGTEFTLI  277. ISSLQPDDFATYYC QQYISYSRT FGQGTKVEIK VL 480 DIQMTQSPSTLSASVGDRVTITCRAS QSISSW LAWYQQKPGKAPKLLIY KAS SLESGVPSRFSGSGSGTEFTLT  278. ISSLQPDDFATYYC QQYISYSRT FGQGTKVEIK VL 490 EIVMTQSPATLSVSPGERGTLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAS TRATGFPARFSGSGSGTEFTLT  279. ISSLQSEDFAVYYC QQYNKWPPFTF GPGTKVDFK VL 500 EIVMTQSPATLSVSPGERGTLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAS TRATGFPARFSGSGSGTEFTLT  280. ISSLQSEDFAVYYC QQYNKWPPFT FGPGTKVDIK VL 504 EIVMTQSPATLSVSPGERATLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAST RATGIPARFSGSGSGTEFTLT 281. ISSLQSEDFAVYYC QQYNKWPPFT FGPGTKVDIK VL 514 DIVMTOFPLSLPVTPGEPASISCRSS QSLLHINEYNY LDWYLKKPGOSPOLLIY LGF NRASGVPDRFSGSGSGT  282. DFTLKISRVEAEDVGVYYC MQALQTPWT LGQGTKVEIK VL 524 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHINEYNY LDWYLKKPGQSPQLLIY LGF NRASGVPDRFSGSGSGT  283. DFTLKISRVEAEDVGVYYC MQALQTPWT LGQGTKVEIK VL 528 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHINEYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGT  284. DFTLKISRVEAEDVGVYYC MQALQTPWT FGQGTKVEIK VL 538 EIVMTQSPATLSVSPGERATLSCRAS QSVSSN VAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLT  285. ISSLQSEDFAVYYC QQYNNWPPFT FGPGTKVDIK VL 548 EIVMTQSPATLSVSPGERATLSCRAS QSVSSN VAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLT  286. ISSLQSEDFAVYYC QQYNNWPPFTF GPGTKVDIK VL 552 EIVMTQSPATLSVSPGERATLSCRAS QSVSSN LAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLT  287. ISSLQSEDFAVYYC QQYNNWPPFT FGPGTKVDIK VL 562 EIVMTQSPATLSVSPGERATLSCRAS QSVSSN VAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLT  288. ISSLQSEDFAVYYC QQYNNWPPFT FGPGTKVDIK VL 572 EIVMTQSPATLSVSPGERATLSCRAS QSVSSN VAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLT  289. ISSLQSEDFAVYYC QQYNNWPPFT FGPGTKVDIK VL 576 EIVMTQSPATLSVSPGERATLSCHAY QSVSSN LAWYQQKPGQAPRLLIY GAS TRATGIPARFSGSGSGTEFTLT 290. ISSLQSEDFAVYYC QQYNNWPPFT FGPGTKVDIK VL 586 DIQLTQSPSFLSASVGDRVTITCWAS QGISSY LAWYQKKPGKAPNLLIY DAS TLQSGVPSRFSGSGSGTEFTLT  291. LSSLQPEDFATYYC QQLNIYPFT FGPGTKVDIK VL 596 DIQLTQSPSFLSASVGDRVTITCWAS QGISSY LAWYQKKPGKAPNLLIY DAS TLQSGVPSRFSGSGSGTEFTLT  292. LSSLQPEDFATYYC QQLNIYPFT FGPGTKVDIK VL 600 DIQLTQSPSFLSASVGDRVTITCRAS QGISSY LAWYQQKPGKAPKLLIY DAS TLQSGVPSRFSGSGSGTEFTLT  293. ISSLQPEDFATYYC QQLNIYPFT FGPGTKVDIK VL 610 EIVLTQSPGTLSLSPGERATLSCRAS QSVSIRYLAWY QQKPGQAPRLLIY GAS SRATGIPDRFSVSVSGTDFTL  294. TITRLEPEDFAVYYC QQYGSSPLT FGGGTKVEIK VL 620 EIVLTQSPGTLSLSPGERATLSCRAS QSVSIRYLAWY QQKPGQAPRLLIY GAS SRATGIPDRFSVSVSGTDFTL  295. TITRLEPEDFAVYYC QQYGSSPLT FGGGTKVEIK VL 624 EIVLTQSPGTLSLSPGERATLSCRAS QSVSIRYLAWY QQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTL  296. TISRLEPEDFAVYYC QQYGSSPLT FGGGTKVEIK VL 634 DVVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGNTY LNWFQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  297. DFTLKISRVEAEDVGVYYC MQGTHWPYT FGQGTKLEIK VL 644 DVVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGNTY LNWFQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  298. DFTLKISRVEAEDVGVYYC MQGTHWPYT FGQGTKLEIK VL 648 DVVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGNTY LNWFQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  299. DFTLKISRVEAEDVGVYYC MQGTHWPYT FGQGTKLEIK VL 658 DVVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGNTY LNWFQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  300. DFTLKISRVEAEDVGVYYC MQGTHWPYT FGQGTKLEIK VL 668 DVVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGNTY LNWFQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  301. DFTLKISRVEAEDVGVYYC MQGTHWPYT FGQGTKLEIK VL 672 DVVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGNTY LNWFQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  302. DFTLKISRVEAEDVGVYYC MQGTHWPYT FGQGTKLEIK VL 682 DVVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGNTY LNWFQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  303. DFTLKISRVEAEDVGVYYC MQGTHWPYT FGQGTKLEIK VL 692 DVVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGNTY LNWFQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  304. DFTLKISRVEAEDVGVYYC MQGTHWPYT FGQGTKLEIK VL 696 DVVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGNTY LNWFQQRPGQSPRRLIY KVS NRDSGVPDRFSGSGSGT  305. DFTLKISRVEAEDVGVYYC MQGTHWPYT FGQGTKLEIK VL 706 KIVLTQSPGTLPLFPGERATLSCRAS QSVNNKF LAWYQQKSGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTL  306. TISGLEPEDFEVYYC QVYGNSLT FGGGTKVEIK VL 716 EIVLTQSPGTLPLFPGERATLSCRAS QSVNNKF LAWYQQKSGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTL 307. TISGLEPEDFEVYYO QVYGNSLTF GGGTKVEIK VL 720 EIVLTQSPGTLSLSPGERATLSCRAS QSVNNKF LAWYQQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTL 308. TISRLEPEDFAVYYC QVYGNSLT FGGGTKVEIK VL 730 DIVMTQFPLSLPVTPGEPASISCRSS QSLLHINEYNY LDWYLKKPGQSPQLLIY LGF NRASGVPDRFSGSGSGT 309. DFTLKISRVEAEDVGVYYC MQALQTPWT FGQGTKVEIK VL 740 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHINEYNY LDWYLKKPGQSPQLLIY LGF NRASGVPDRFSGSGSGT 310. DFTLKISRVEAEDVGVYYC MQALQTPWT FGQGTKVEIK VL 744 DIVMTQSPLSLPVTPGEPASISCRSS QSLLHINEYNY LDWYLQKPGQSPQLLIY LGS NRASGVPDRFSGSGSGT 311. DFTLKISRVEAEDVGVYYC MQALQTPWT FGQGTKVEIK GROUP E VH 508.20 EVQLVESGGGLVQPGGSLRLSCAAS GFTFTGYAIH WVRQAPGKGLEWVG RISPANGNTNYADSVKG RFTISADT  312. SKNTAYLQMNSLRAEDTAVYYCAR WIGSRELYIMDY WGQGTLVTVSS VH 508.04 EVQLVESGGGLVQPGGSLRLSCAAS GFTFTGYAIH WVRQAPGKGLEWVG RISPAHGNTNYADSVKG RFTISADT  313. SKNTAYLQMNSLRAEDTAVYYCAR WIGSRELYIMDY WGQGTLVTVSS VH 508.06 EVQLVESGGGLVQPGGSLRLSCAAS GFTFTGYAIH WVRQAPGKGLEWVG RISPANGNTNYADSVKG RFTISADT  314. SKNTAYLQMNSLRAEDTAVYYCAR WIGSRELYIMDY WGQGTLVTVSS VH 508.28 EVQLVESGGGLVQPGGSLRLSCAAS GFTFTRHTIH WVRQAPGKGLEWVG RISPANGNTNYADSVKG RFTISADT  315. SKNTAYLQMNSLRAEDTAVYYCAR WIGSRELYIMDY KGQGTLVTVSS VH 508.33 EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSTAIH WVRQAPGKGLEWVG RISPANGNTNYADSVKG RFTISADT  316. SKNTAYLQMNSLRAEDTAVYYCAR WIGSRELYIMDY WGQGTLVTVSS VH 508.84 EVQLVESGGGLVQPGGSLRLSCAAS GFTFTGYAIH WVRQAPGKGLEWVG RISPANGNTNYADSVKG RFTISADT 317. SKNTAYLQMNSLRAEDTAVYYCAR WIGSRELYIMDY WGQGTLVTVSS VL 508.20 DIQMTQSPSSLSASVGDRVTITC RASQDVSSAVA WYQQKPGKAPKLLIY SASSLYS GVPSRFSGSRSGTDFTLT 318. ISSLQPEDFATYYC QQSYTTPPT FGQGTKVEIKR VL 508.04 DIQMTQSPSSLSASVGDRVTITC RASQDVSSAVA WYQQKPGKAPKLLIY SASSLYS GVPSRFSGSRSGTDFTLT 319. ISSLQPEDFATYYC QQSYPAPAT FGQGTKVEIKR VL 508.06 DIQMTQSPSSLSASVGDRVTITC RASQDVSSAVA WYQQKPGKAPKLLIY SASSLYS GVPSRFSGSRSGTDFTLT 320. ISSLQPEDFATYYC QQSYPSPAT FGQGTKVEIKR VL 508.28 DIQMTQSPSSLSASVGDRVTITC RASQDVSSAVA WYQQKPGKAPKLLIY SASSLYS GVPSRFSGSRSGTDFTLT 321. ISSLQPEDFATYYC QQSYRIQPT FGQGTKVEIKR VL 508.33 DIQMTQSPSSLSASVGDRVTITC RASQDVSSAVA WYQQKPGKAPKLLIY SASSLYS GVPSRFSGSRSGTDFTLT 322. ISSLQPEDFATYYC QQSYRIQPT FGQGTKVEIKR VL 508.84 DIQMTQSPSSLSASVGDRVTITC RASQDVSSAVA WYQQKPGKAPKLLIY SASSLYS GVPSRFSGSRSGTDFTLT  323. ISSLQPEDFATYYC QQSYPAPST FGQGTKVEIKR GROUP F VH EVQLVESGGGLVKPGGSLRLSCAAS GFPFSKLGMV WVRQAPGKGLEWVS TISSGGGYTYYPDSVKG RFTISRDN  324. AKNSLYLQMNSLRAEDTAVYYCAR EGISFQGGTYTYVMDY WGQGTLVTVSS VL DIVMTQSPLSLPVTPGEPASISC RSSKSLLHRNGITYSY WYLQKPGQSPQLLIY QLSNLAS GVPDRFSGSGSGT  325. DFTLKISRVEAEDVGVYYC YQNLELPLT FGQGTKVEIK GROUP G VH IB20 QVQLVQSGAEVKKPGESLKISCKGS GYSFTNYWIS WVRQMPGKGLE WMGIIYPGDSYTNYSPSFQG QVTISADK  326. SISTAYLQWSSLKASDTAMYYCAR DYWYKPLFDI WGQGTLVTVSS VL IB20 DIVMTQSPDSLAVSLGERATINC RSSQSVLYSSNNKNYL AWYQQKPGQPPK LLIYWASTRES GVPDRFSGSGSG  327. TDFTLTISSLQAEDVAVYYC QQYSSFPI TFGQGTKVEIKR IB20 Variant  GYSFTX₁YX₂IX₃  328. HC CDR1 X₁ = N or D  X₂ = W or Y  IB20 Variant  WMGX₁IYPGDSX₂TX₃YX₄X₅X₆FQG  329. HC CDR2 X₁ = I, R, W, L, or M  X₂ = Y or D  X₃ = N, R, H, S, K, Q  X₄ = S or N  X₅ = P, Q, H  X₆ = S, K, N, or R IB20 Variant  DX₁X₂X₃X₄X₅X₆X₇DX₈ 330. HC CDR3 X₁ = Y, R, or H  X₂ = W, Y, G, A, or F  X₃ = Y or S  X₄ = K, R, T, G, S, D, E, H, or N X₅ = P, S, G, D, A, H, or Y X₆ = L, Y, F, A, D, H, P, or S X₇ = F or S  X₈ = I, V, Y, F, or N  IB30 Variant  WMGX₁IYPGDSYTX₂YSX₃SFQG 331. VH CDR2  X₁ = I or R  X₂ = N, R, H, or S  X₃ = P or Q  IB30 Variant DYWYX₁X₂X₃FDX₄ 332. VH CDR3  X₁ = K, G, R, S, D, E, H,N, or Q X₂ = P, G, D, S, A, H, R, or Y  X₃ = L, A, Y, D, F, H, P, S, or V X₄ = I, Y, F, or N  IB20 Variant X₁ Ser Ser Gln Ser Val X₂ X₃ Ser X₄ X₅ X₆ Lys Asn X₇ Leu X₈ 333. LC CDR1  X₁ = Arg, Lys, His, Asn, Gln or Ser X₂ = Leu or Phe  X₃ = Tyr or His  X₄ = Ser, Arg or Gly  X₅ = Asn or Thr  X₆ = Asn, Arg, His or Ser  X₇ = Tyr or Phe  X₅ = Ala or Thr IB20 Variant LLIYX₁X₂SX₃RX₄X₅ 334. LC CDR2 X₁ = Trp, Phe or Leu X₂ = Ala or Thr X₃ = Thr, Ile, Ala or val X₄ = Glu, Ala or Lys X₅ = Ser or Thr IB20 Variant QQYX₁X₂X₃PX₄ 335. LC CDR3 X₁ = Ser or Tyr  X₂ = Ser or Thr  X₃ = Phe, Tyr, Leu, Thr, His, Ile, Asn, Pro or Ser  X₄ = Ile, Arg, Val, Tyr, Asp, Phe, Gly, His, Leu, Asn or Ser 1B20 Antibody  RSSQSVLYSSNNKNX₁LX₂  336. Variant VL  X₁ = Tyr or Phe  CDR1 Sequence X₂ = Ala or Thr 1B20 Antibody  LLIYX₁ASTRX₂X₃ 337. Variant VL  X₁ = Trp, Phe or Leu  CDR2 Sequence X₂ = Glu or Lys  X₃ = Ser or Thr 1B20 Antibody  QQYSSX₁PX₂  338. Variant VL  X₁ = Phe, Tyr, Thr, Ile, Asn or Scr  CDR3 Sequence X₂ = Ile, Tyr, Arg, Phe, His, Leu, Asn or Ser VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTX₁YX₂IX₃WVRQMPGKGLEWMGX₄IYPGDSX₅TX₆YX₇X₈X₉F VARIANT  QGQVTISADKSISTAYLQWSSLKASDTAMYYCARDX₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆DX₁₇WGQGTLVTVSS  SEQUENCE X₁ = Asn or Asp  X₂ = Trp or Tyr  X₃ = Ser, Thr or Ala  X₄ = Ile, Arg, Trp, Leu or Met  X₅ = Tyr or Asp  X₆ = Asn, Arg, His, Ser, Lys or Gln  X₇ = Ser or Asn  X₈ = Pro, Gln or His  X₉ = Ser, Lys, Asn or Arg  X₁₀ = Tyr, Arg or His  X₁₁ = Trp, Tyr or Gly  339. X₁₂ = Tyr or Ser X₁₃ = Lys, Arg, Thr, Gly, Ser, Asp, Glu, His, Asn or Gln X₁₄ = Pro, Ser, Gly, Asp, Ala, His, Arg or Tyr X₁₅ = Leu, Tyr, Phe, Ala, Asp, His, Pro, Ser or Val X₁₅ = Phe or Ser X₁₇ = Ile, Val, Tyr, Phe or Asn VH; 1B20 QVQLVQSGAEVKKPGESLKISCKGSGYSFTX₁YX₂IX₃WVRQMPGKGLEWMGX₄IYPGDSYTX₅YSX₆SFQGQ 340. VARIANT VTISADKSISTAYLQWSSLKASDTAMYYCARDWYWX₇X₈X₉FDX₁₀WGQGTLVTVSS SEQUENCE X₁ = Asn or Asp X₂ = Trp or Tyr X₃ = Ser, Thr or Ala X₄ = Ile or Arg X₅ = Asn, Arg, His, Ser, Lys or Gln X₆ = Pro or Gln X₇ = Lys, Gly, Arg, Ser, Asp, Glu, His, Asn or Gln X₈ = Pro, Gly, Asp, Ser, Ala, His, Arg or Tyr X₉ = Leu, Ala, Tyr, Asp, Phe, His, Pro, Ser or Val X₁₀ = Ile, Tyr, Phe or Asn VL; 1B20 DIVWQSPDSLAVSLGERATINCX₁SSQSVX₂X₃SX₄X₅X₆KNX₇LX₈WYQQKPGQPPKLLIYX₉X₁₀SX₁₁RX₁₂ 341. Variant X₁₃GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYX₁₄X₁₅X₁₆PX₁₇TFGQGTKVEIKR Sequence X₁ = Arg, Lys, His, Asn, Gln or Ser X₂ = Leu or Phe X₃ = Tyr or His X₄ = Ser, Arg or Gly X₅ = Asn or Thr X₆ = Asn, Arg, His or Ser X₇ = Tyr or Phe X₈ = Ala or Thr X₉ = Trp, Phe or Leu X₁₀ = Ala or Thr X₁₁ = Thr, Ile, Ala or Val X₁₂ = Glu, Ala or Lys X₁₃ = Ser or Thr X₁₄ = Ser or Tyr  X₁₅ = Ser or Thr  X₁₆ = Phe, Tyr, Leu, Thr, His, Ile, Asn, Pro or Ser  X₁₇ = Ile, Arq, Val, Tyr, Asp, Phe, Gly, His, Leu, Asn or Ser VL; 1B20  DIVMTQSPDSLAVSLGERATINCRSSQSVLYSSNNKNX₁LX₂WYQQKPGQPPKLLIYX₃ASTRX₄X₅GVPDRFSGS 342. VARIANT  GSGTDFTLTISSLQAEDVAVYYCQQYSSX₆PX₇TFGQGTKVEIKR  SEQUENCE X₁ = Y or F  X₂ = A or T  X₃ = W, F, or L  X₄ = E or K  X₅ = S or T  X₆ = F, Y, T, I, N, or S  X₇ = I, Y, R, F, H, L, N, or S  VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGLIYPGDSYTRY 343. Variant  NPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYYSRPFSDIWGQGTLVTVSS Sequence F120 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGLIYPGDSYTRY 344. Variant  NPKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYYSRPYSDVWGQGTLVTVSS Sequence F116 VH; 1B20  QVQLVQSGAEVKKPGESLKTSCKGSGYSFTDYYISWVRQMPGKGLEWMGLIYPGDSYTRY 345. Variant  SPNFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHGYRPYSDIWGQGTLVTVSS Sequence F119 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGLIYPGDSYTNY 346. Variant  NPNFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHYSTPFFDVWGQGTLVTVSS Sequence F113 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGLIYPGDSYTRY 347. Variant  NRKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYYSRPLSDVWGQGTLVTVSS Sequence E2 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGLIYPGDSYTRY 348. Variant  SPRFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHGYKPYSDIWGQGTLVTVSS Sequence G4 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGLIYPGDSYTRY 349. Variant  SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYYSKPLFDVWGQGTLVTVSS Sequence F4 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGIIYPGDSYIHY 350. Variant  NQNFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYYSRPFSDIWGQGTLVTVSS Sequence B9 VH; 1B20  QVKLVQSGAEVKKPGESLKISCKGSGYSFTNYYIAWVRQMPGKGLEWMGIIYPGDSYTHY 351. Variant  NPKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHGYKPFSDIWGQGTLVTVSS Sequence C3 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIAWVRQMPGKGLEWMGVIYPGDSYTRY 352. Variant  NPKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWSRPYFDIWGQGTLVTVSS Sequence F2 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIAWVRQMPGKGLEWMGIIYPGDSYTHY 353. Variant  NPKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWSRPFSDVWGQGTLVTVSS Sequence F7 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGIIYPGDSYTRY 354. Variant  NPNFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWSKPLSDVWGQGTLVTVSS Sequence A7 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGLIYPGDSYTHY 355. Variant  NPNFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWSKPYFDVWGQGTLVTVSS Sequence G8 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYITWVRQMPGRGLEWMGIIYPGDSYTRY 356. Variant  NPKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWSKPFSDVWGQGTLVTVSS Sequence H4 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYITWVRQMPGKGLEWMGLIYPGDSYTRY 357. Variant  SPRFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWSRPYSDVWGQGTLVTVSS Sequence D5 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYIAWVRQMPGKGLEWMGIIYPGDSYTRY 358. Variant  NPKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWSRPFSDVWGQGTLVTVSS Sequence D4 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGLIYPGDSYTHY 359. Variant  NPNFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWSKPLFDVWGQGTLVTVSS Sequence B4 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYIAWVRQMPGKGLEWMGIIYPGDSYTRY 360. Variant  NPRFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWSKPLSDIWGQGTLVTVSS Sequence H1 VH; 1B20  QVKLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGIIYPGDSYTHY 361. Variant  NPKFQCQVTISADKSISTAYLQWSSLKASDTAMYYCARDRWSKPLFDVWCQCTLVTVSS Sequence G2 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGLIYPGDSYTRY 362. Variant  NPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWSKPLSDIWGQGTLVTVSS Sequence A1 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGSIYPGDSYTHY 363. Variant  SHKFQGQVTISADKSISTAYLQWSSLKASDTAIYYCARDHWSRPFFDVWGQGTLVTVSS Sequence A4 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYIAWVRQMPGKGLEWMGLIYPGDSYTSY 364. Variant  NPRFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWSTPYFDIWGQGTLVTVSS Sequence C2 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGIIYPGDSYTSY 365. Variant  SPRFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWSRPLFDVWGQGTLVTVSS Sequence H5 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGLIYPGDSYTHY 366. Variant  NPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWYKPFSDIWGQGTLVTVSS Sequence F6 VH; 1B20 QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGIIYPGDSYTNY 367. Variant  NPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWYRPFSDIWGQGTLVTVSS Sequence B6 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGIIYPGDSYTHY 368. Variant  SPNFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWYTPFFDVWGQGTLVTVSS Sequence B1 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGLIYPGDSYTNY 369. Variant  SPKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWSKPLSDVWGQGTLVTVSS Sequence F1 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGMIYPGDSYTHY 370. Variant  SPNFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWSKPYFDVWGQGTLVTVSS Sequence A8 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGMIYPGDSYTSY 371. Variant  NPKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWSKPYFDIWGQGTLVTVSS Sequence B3 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGMIYPGDSYTNY 372. Variant  NQKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWSRPYSDIWGQGTLVTVSS Sequence F8 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGMIYPGDSYTHY 373. Variant  SQNFQGQVTTSADKSTSTAYLQWSSLKASDTAMYYCARDHWSRPFFDVWGQGTLVTVSS Sequence H8 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGMIYPGDSYTRY 374. Variant  SPSFQGQVTISADKSISTAYLQSSSLKASDTAMYYCARDYWYKPFSDVWGQGTLVTVSS Sequence B5 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGMIYPGDSYTSY 375. Variant  NPNFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWSKPFFDIWGQGTLVTVSS Sequence E1 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYITWVRQMPGKGLEWMGMIYPGDSYTRY 376. Variant SPKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWSKPLSDVWGQGTLVTVSS Sequence E8 VH; IB20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGMIYPGDSYTRY 377. Variant  SPKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWYRPYSDIWGQGTLVTVSS Sequence C1 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYITWVRQMPGKGLEWMGMIYPGDSYTHY 378. Variant  SQRFQGQVTISADKSISTAYLQWSSLKASDTAIYYCARDHWSRPLFDVWGQGTLVTVSS Sequence H3 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGMIYPGDSYTRY 379. Variant  SPKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWYRPYSDIWGQGTLVTVSS Sequence A9 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGWIYPGDSYTHY 380. Variant  SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWSKPYFDVWGQGTLVTVSS Sequence G7 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYITWVRQMPGKGLEWMGMIYPGDSYTHY 381. Variant  NPSFQCQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWYKPLFDIWCQCTLVTVSS Sequence C6 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYINWVRQMPGKGLEWMGMIYPGDSYTNY 382. Variant  NPKFQGQVTISADKSISTAYLQWSSLKASDTAIYYCARDHWSRPFSDVWGQGTLVTVSS Sequence G6 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGWIYPGDSYTSY 383. Variant  NQKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWSKPYSDVWGQGTLVTVSS Sequence E4 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYITWVRQMPGKGLEWMGMIYPGDSYTNY 384. Variant  RHNFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWSRPYFDIWGQGTLVTVSS Sequence F5 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGWIYPGDSYTHY 385. Variant  SHNFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWSRPFSDVWGQGTLVTVSS Sequence C7 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQXPGKGLEWMGMIYPCOSYTSY 386. Variant SPRFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWYRPFSDIWGQGTLVTVSS Sequence E3 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYITWVRQMPGKGLEWMGMIYPGDSYTRY 387. Variant  SPKFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWSKPFFDVWGQGTLVTVSS Sequence D3 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGMIYPGDSYTHY 388. Variant  SQSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWYRPLFDIWGQGTLVTVSS Sequence D8 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIAWVRQMPGKGLEWMGMIYPGDSYTSY 389. Variant  SHRFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWYRPYFDIWGQGTLVTVSS Sequence C8 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGWIYPGDSYTHY 390. Variant  NPNFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDRWSTPYFDVWGQGTLVTVSS Sequence E5 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYISWVRQMPGKGLEWMGMIYPGDSYTHY 391. Variant  SPNFQCQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWSKPYFDVWCQCTLVTVSS Sequence B8 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGPEWMGMIYPGDSYTRY 392. Variant  SQKFQGQVTISADKSISTAYLQWSSLKASDTAIYYCARDHWSRPLSDIWGQGTLVTVSS Sequence H7 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGWIYPGDSYTHY 393. Variant  NPMFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDRWSRPYFDIWGQGTLVTVSS Sequence A5 VH; 1B20  QVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYISWVRQMPGKGLEWMGMIYPGDSYTRY 394. Variant  SPNFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDHWYRPLSDIWGQGTLVTVSS Sequence A3 1B20 Antibody  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWISWVRQMPGKGLEWMGIIYPGDSYTKY 395. Variant VH  SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWYKPLFDIWGQGTLVTVSS Sequence N59K 1B20 Antibody QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWISWVRQYPGKCLEWMGIIYPGDSYTQY 396. Variant VH  SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWYKPLFDIWGQGTLVTVSS Sequence N59Q 1B20 Antibody  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWISWVRQMPGKGLEWMGIIYPGDSYTRY 397. Variant VH  SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYWYKPLFDIWGQGTLVTVSS Sequence N59R 1B20 Antibody  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWISWVRQMPGKGLEWMGIIYPGDSYTNY 398. Variant VH  SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYAYKPLFDIWGQGTLVTVSS Sequence  W101A 1B20 Antibody  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWISWVRQMPGKGLEWMGIIYPGDSYTNY 399. Variant VH  SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYFYKPLFDIWGQGTLVTVSS Sequence  W101F 1B20 Antibody  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWISWVRQMPGKGLEWMGIIYPGDSYTNY 400. Variant VH  SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARDYYYKPLFDIWGQGTLVTVSS Sequence  W101Y GROUP H Linker  GGGGGGGGGGGCGG 401. Sequence  (used in  DSB#1) Linker  GGGGGGGGGGGGCG 402. Seqeunce (used in  DSB#2) Linker GGGGSGGGGS 403. Group I PC9#3  EVQLVESGGGLVQPGGSLRLSCAASGFTFNNYAMNWVRQAPGKGLDWVSTISGSGGTTNYADSVKGRFIISRDS 404. variable  SKHTLYLQMNSLRAEDTAVYYCAKDSNWGNFDLWGRGTLVTVSS heavy chain PC9#3  DIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNRNFLGWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSG 405. variable  TDFTLTISSLQAEDVAVYYCQQYYTTPYTFGQGTKLEIK light chain PC9#4  EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYISYADSVKGRFTISRDN 406. variable  AKNSLYLQMNSLRAEDTAVYFCARDYDFWSAYYDAFDVWGQGTMVTVSS heavy chain PC9#4  QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLISGNSNRPSGVPDRFSGSKSGTSAS 407. variable  LAITGLQAEDEADYYCQSYDSSLSGSVFGGGTKLTVL light chain PC9#5  EMQLVESCCCLVQPCCSLRLSCAASCFTFSSHWMKWVRQAPCKCLEWVANINQDCSEKYYVDSVKCRFTISRDN 408. variable  AKNSLFLQMNSLRAEDTAVYYCARDIVLMVYDMDYYYYGMDVWGQGTTVTVSS heavy chain PC9#5  DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGNNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGT 409. variable  DFTLKISRVEAEDVGVYYCMQTLQTPLTFGGGTKVEIK light chain PC9#6  QVQLVQSGAEVKKPGSSVKVSCKASGGTFNSHAISWVRQAPGQGLEWMGGINPILGIANYAQKFQGRVTITADE 410. variable  STSTAYMELSSLRSEDTAVYYCARHYEIQTGRYGMNVYYLMYRFASWGQGTLVTVSS heavy chain PC9#6  DIQMTQSPSSLSASVGDRVTITCRASQGIRSALNWYQQKPGKAPKLLIYNGSTLQSGVPSRFSGSGSGTDFTLT 411. variable  ISSLQPEDFAVYYCQQFDGDPTFGQGTKVEIK light chain PC9#7  QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWISWVRQMPGKGLEWMGIIYPGDSYTNYSPSFQGQVTISADK 412. variable  SISTAYLQWSSLKASDTAMYYCARDYWYKPLFDIWGQGTLVTVSS heavy chain PC9#7  DIVMTQSPDSLAVSLGERATINCRSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSG 413. variable  TDFTLTISSLQAEDVAVYYCQQYSSFPITFGQGTKVEIK light chain NIP228 QVNLRESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGEGLEWVSAISGSGGSTYYADSVKGRFTISRDN 414. variable  SKNTLYLQMXSLRAEDTVYYCAKRFGEFAFDIGRGTTVTVSS heavy chain NIP228  AIRMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLT 415. variable  ISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK light chain Human IgG4  ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR 416. Fc fragment EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS  LSLSLG Human  QVQLVQSGAEVKKPGASVfCVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDT 417. germline  STSTVYMELSSLRSEDTAVYYCARD 1-46 (DP-7) Human  DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFT 418. germline VK1  ISSLQPEDIATYYCQQ O18 O8 (DPK1) HS9_DSB7_  HGEGTFTSDLSKQMEEECARLFIEWLKNGGPSSGAPPPGCGGGGGSGGGGSADIQMTQSPSSLSASVGDRVTIT 419. V19A_L2 CQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWR TFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HS9_DSB7_  HGEGTFTSDLSKQMEEECARLFIEWLKNGGPSSGAPPPGCGGGGGSADIQMTQSPSSLSASVGDRVTITCQASQ 420. V19A_L1 DVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRTFGQG TKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC HS9_DSB7_  HGEGTFTSDLSKQMEEECARLFIEWLKNGGPSSGAPPPGCGADIQMTQSPSSLSASVGDRVTITCQASQDVKTA 421. V19A_L0 VAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2.7A4_VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYSMNWVRQAPGKGLEWVSSISSSGDYIYYADSVKGRFTISRDN  422. AKNSLYLQMNSLRAEDTAVYYCARDLVTSMVAFDYWGQGTLVTVSS 2.7A4_VL SYELTQPPSVSVSPGQTARITCSGDALPQKYVFWYQQKSGQAPVLVIYEDSKRPSGIPERFSGSSSGTMATLTI 423. SGAQVEDEADYYCYSTDRSGNHRVFGGGTKLTVL GROUP J HS1_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEIKPSGGSTSYNQKFQGRVTMTRDTSTST 424. VYMELSSLRSEDTAVYYCARERPLYASDLWGQGTTVTVSS HS1_VL DIQMTQSPSSLSASVGDRVTITCQASQDVYTAVAWYQQKPGKAPKLLIY YASYRYTGVPSRFSGSGSGTDFTFTISSL 425. QPEDIATYYCQQRYSLWRTFGQGTKLEIK HS2_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEIKPSGGSTSYNQKFQGRVTMTRDTSTST 426. VYMELSSLRSEDTAVYYCARERPLYASDLWGQGTTVTVSS HS2_VL DIQMTQSPSSLSASVGDRVTITCQASQDVYTAVAWYQQKPGKAPKLLIY SASYRYTGVPSRFSGSGSGTDFTFTISSL 427. QPEDIATYYCQQRYSLWRTFGQGTKLEIK HS3_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEIKPSGGSTSYNQKFQGRVTMTRDTSTST 428. VYMELSSLRSEDTAVYYCARERPLYASDLWGQGTTVTVSS HS3_VL DIQMTQSPSSLSASVGDRVTITCQASQDVKTAVAWYQQKPGKAPKLLIY YASYRYTGVPSRFSGSGSGTDFTFTISSL 429. QPEDIATYYCQQRYSLWRTFGQGTKLEIK HS4_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEIKPSGGSTSYNQKFQGRVTMTRDTSTST 430. VYMELSSLRSEDTAVYYCARERPLYASDLWGQGTTVTVSS HS4_VL DIQMTQSPSSLSASVGDRVTITCQASQDVKTAVAWYQQKPGKAPKLLIY SASYRYTGVPSRFSGSGSGTDFTFTISSL 431. QPEDIATYYCQQRYSLWRTFGQGTKLEIK HS5_VH QVQLVQSGAKVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEIKPSGGSTSYNQKFQGRVTMTRDTSTST 432. VYMELSSLRSEDTAVYYCARERPLYASDLWGQGTTVTVSS HS5_VL DIQMTQSPSSLSASVGDRVTITCQASQDVSTAVAWYQQKPGKAPKLLIY SASYRYTGVPSRFSGSGSGTDFTFTISSL 433. QPEDIATYYCQQRYSLWRTFGQGTKLEIK HS6_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEISPSGGSTSYNQKFQGRVTMTRDTSTST 434. VYMELSSLRSEDTAVYYCARERPLYASDLWGQGTTVTVSS HS6_VL DIQMTQSPSSLSASVGDRVTITCQASQDVYTAVAWYQQKPGKAPKLLIY YASYRYTGVPSRFSGSGSGTDFTFTISSL 435. QPEDIATYYCQQRYSLWRTFGQGTKLEIK HS7_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEISPSGGSTSYNQKFQGRVTMTRDTSTST 436. VYMELSSLRSEDTAVYYCARERPLYASDLWGQGTTVTVSS HS7_VL DIQMTQSPSSLSASVGDRVTITCQASQDVYTAVAWYQQKPGKAPKLLIY SASYRYTGVPSRFSGSGSGTDFTFTISSL 437. QPEDIATYYCQQRYSLWRTFGQGTKLEIK HS8_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEISPSGGSTSYNQKFQGRVTMTRDTSTST 438. VYMELSSLRSEDTAVYYCARERPLYASDLWGQGTTVTVSS HS8_VL DIQMTQSPSSLSASVGDRVTITCQASQDVKTAVAWYQQKPGKAPKLLIY YASYRYTGVPSRFSGSGSGTDFTFTISSL 439. QPEDIATYYCQQRYSLWRTFGQGTKLEIK HS9_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEISPSGGSTSYNQKFQGRVTMTRDTSTST 440. VYMELSSLRSEDTAVYYCARERPLYASDLWGQGTTVTVSS HS9_VL DIQMTQSPSSLSASVGDRVTITCQASQDVKTAVAWYQQKPGKAPKLLIY SASYRYTGVPSRFSGSGSGTDFTFTISSL 441. QPEDIATYYCQQRYSLWRTFGQGTKLEIK HS10_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGEISPSGGSTSYNQKFQGRVTMTRDTSTST 442. VYMELSSLRSEDTAVYYCARERPLYASDLWGQGTTVTVSS HS10_VL DIQMTQSPSSLSASVGDRVTITCQASQDVSTAVAWYQQKPGKAPKLLIY SASYRYTGVPSRFSGSGSGTDFTFTISSL 443. QPEDIATYYCQQRYSLWRTFGQGTKLEIK HS9_DS  HYEGTFTSDLSKQMEEECVRLFIEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT  444. B7_G2Y ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HVEGTFTSDLSKQMEEECVRLFIEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT  445. B7_G2V ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLOPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HTEGTFTSDLSKQMEEECVRLF1EWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT  446. B7_G2T ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HQEGTFTSDLSKQMEEECVRLFIEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT  447. B7_G2Q ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLOPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HNEGTFTSDLSKQMEEECVRLFISWLKNGGPSSGAPPLGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 448. B7_G2N ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HIEGTFTSDLSKQMEEECVRLFIEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTOSPSSLSASVGDRVT 449. B7_G2I ITCQASQDVKTAVAWYQQKPGKAPKLLTYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HFEGTFTSDLSKQMEEECVRLF1EWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 450. B7_G2F ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDTATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMGEECVRLFIEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 451. B7_E15G ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMAEECVRLFIEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 452. B7_E15A ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECTRLFIEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADTQMTQSPSSLSASVGDRVT 453. B7_V19T ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECSRLFIEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 454. B7_V19S ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTPTSDLSKQMEEECGRLFIEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 455. B7_V19G ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT HS9_DS  HGEGTFTSDLSKQMEFECARLFIEKLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 456. B7_V19A ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9.DS  HGEGTFTSDLSKQMEEECVRLFTEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT  457. B7J23T ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECVRLFSEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 458. B7_I23S ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECVRLFGEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 459. B7_L23G ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECVRLFAEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 460. B7_L23A ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECVRLFIEWTKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 461. B7_L26T ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECVRLFIEWSKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSA5VGDRVT 462. B7_L26S ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECVRLFIEWPKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQ3PSSLSASVGDRVT 463. B7_L26P ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT HS9_DS  HGEGTFTSDLSKQMEEECVRLFIEWNKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 464. B7_L26N ITCQASQDVKTAVAWYQQKPGKAPKLLIYSA5YRYTGVP5RFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECVRLFTEWQKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT  465. B7_L26Q ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECVRLFIEWMKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 466. B7_L26M ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECVRLFIEWIKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 467. B7_L26I ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9JDS  HGEGTFTSDLSKQMEEECVRLFIEWHKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 468. B7_L26H ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECVRLFIEWGKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 469. B7_L26G ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECVRLFIEWEKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 470. B7_L26B ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTTSSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECVRLFIEMDKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 471. B7_L26D ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK HS9_DS  HGEGTFTSDLSKQMEEECVRLFIEWLKNGGPSSGAPPPGCGGGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRVT 472. B7 ITCQASQDVKTAVAWYQQKPGKAPKLLIYSASYTYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQRYSLWRT  FGQGTKLEIK GLP-1  HGEGTFTSDVSSYLEEQAAKEFIAWLVKGG 473. analogue NGS#1 HGEGTFTSDVSSYLEEQ N A S EFIAWLVKGG 474. NGS#2 HGEGTFTSDVSSYLEEQAAKEFIAWLV N G S 475. NGS#3 HGEGTFTSDVSSYLEEQAAKEFIAWLVK N G 476. NGS#4 HGEGTFTSDVSSYLEEQAAKEFIAWLVKG N 477. NGS#5 HGEGTFTSDVSSYLEEQAAKEFIAWLVKGG 478. NGS#6 HGEGTFTSDVSSYLEEQAAKEFIAWLVKGG 479. NGS#7 HGEGTFTSDVSSYLEEQAAKEFIA N L S KGG 480. NGS#8 HGEGTFTSDVSSYLEEQAAKEFIA N L T KGG 481. Linker GGGGGSGGGGSGGGGSA 482. Linker S GGGGSGGGGSGGGGSA 483. Linker G S GGGSGGGGSGGGGSA 484. Linker N G S GGSGGGGSGGGGSA 485. Linker G N G S GSGGGGSGGGGSA 486. PC9_2_DSB#1

GGGG 487. VL  SGGGGSGGGGSADIQMTQSPSSLSASVGDRVTITC KASQDVHTAVA WYQQKPGKAPKLLIY HASYRYT GVPSRFSGSG (Version B)  SGTDFTFTISSLQPEDIATYYC QQRYSLWRT FGQGTKLEIK (with K at  end of  variable  light chain) PC9_2_ DSB#3 

GGGGSGGGGSGGGGSADIQMTQSP 488. VL  SSLSASVGDRVTITC KASQDVHTAVA WYQQKPGKAPKLLIY HASYRYT GVPSRFSGSGSGTDFTFTISSLQPEDIAT (Version B)  YYC QQRYSLWRT FGQGTKLEIK  (with K at  end of  variable  light chain) PC9_2_NGS#7 

GGGGSGGGGSGGGGSADIQMTQSPSSLSASVGDRV 489. VL (Version  TITC KASQDVHTAVA WYQQKPGKAPKLLIY HASYRYT GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQRYSLWR B) (with K at T FGQGTKLEIK end of  variable  light chain) PC9_2_DSB#7 

GGGGSGGGGSGGGGSADIQM 490. VL (Version  TQSPSSLSASVGDRVTITC KASQDVHTAVA WYQQKPGKAPKLLIY HASYRYT GVPSRFSGSGSGTDFTFTISSLQPE B) (with K at  DIATYYC QQRYSLWRT FGQGTKLEIK end of  variable  light chain) Engineered  EVQLVE3GGGLVKPGGSLRL3CAASGFPFSKLGMVWVRQAPGKGLEWVSTISSGGGYTYYPDSVKGRFTISRDNAKN  PCSK9  SLY 491. antibody VH LQMNSLRAEDTAVYYCAREGISFQGGTYTYVMDYWGQGTLVTVSS Engineered  DIVMTQSPLSLPVTPGEPASISCRSSKSLLHRNGITYSYWYLQKPGQSPQLLIYQLSNLASGVPDRFSGSGSGTDFT  PCSK9  LKI 492. antibodv VL SRVEAEDVGVYYCYQNLELPLTFGQGTKVEIK Engineered  GFPFSKLGMV 493. PCSK9  antibody  VH CDR1 Engineered  TISSGGGYTYYPDSVK 494. PCSK9  antibody  VH CDR2 Engineered  EGISFQGGTYTYVMDY 495. PCSK9  antibodv  VH CDR3 Engineered  RSSKSLLHRNGITYSY 496. PCSK9  antibody  VL CDR1 Engineered  QLSNLAS 497. PCSK9  antibody  VL CDR2 Engineered  YQNLELPLT 498. PCSK9  antibody  VL CDR3

DESCRIPTION OF THE EMBODIMENTS I. Anti-PCSK9˜GLP-1 Fusion Molecules

The present disclosure is directed to fusions of antibodies (e.g., anti-PCSK9 antibodies or antigen-binding fragments thereof) with a GLP-1 moiety.

In one embodiment the fusion is constructed as:

-   -   GLP-1 moiety—Linker—Antibody Light Chain.

or

-   -   GLP-1 moiety-Linker-Antibody Heavy Chain

The fusion protein can be constructed as a genetic fusion. Alternatively, the fusion protein may be constructed as a chemical conjugate, such as through a cysteine:cysteine disulfide bond.

Other arrangements of the GLP-1 moiety and antibody portion are also within the scope herein.

A. Antibodies and Antigen-Binding Fragments Thereof

1. Anti-PCSK9 Portion

The antibody portion may be an anti-PCSK9 antibody or an antigen-binding fragment thereof. In one embodiment, the anti-PCSK9 portion provides an LDLc (bad cholesterol) lowering effect.

In one embodiment, the anti-PCSK9 VL portion may be SEQ ID NO: 2 (PC9_2_HS9). In another embodiment, the anti-PCSK9 VL portion may be an antigen binding portion of SEQ ID NO: 2. In one embodiment, the anti-PCSK9 VL portion may comprise all six CDRs of SEQ ID NO: 2. In one embodiment, the anti-PCSK9 VH portion may be a pH dependent version of the antibody, as shown in SEQ ID NO: 5.

In one embodiment, the antibody or antigen-binding fragment thereof may be pH dependent, such that the antibody binding to the antigen is pH dependent. This can be used to modify half-life of the antibody and/or antigen. By modifying the antibody half-life, in one embodiment, we mean lengthening the half-life. By modifying the antibody half-life, in one embodiment, we mean shortening the half-life. In one embodiment, the antibody half-life may be modified (lengthened or shortened) to maximize the stability of it fusion partner (i.e., GLP-1). By modifying the antigen half-life we may also mean that the antigen-antibody complex can change the antigen's half-life, such as through antibody-mediated degradation (shortening the half-life) or by protecting the antigen from the typical degradation process (lengthening the half-life). In one instance, antibodies may have a higher affinity for the antigen at pH 7.4 as compared to endosomal pH (i.e., pH 5.5-6.0), such that the K_(D) ratio at pH 5/5/pH 7.4 or at pH 6.0/pH 7.2 is 2 or more. Methods of engineering pH dependent antibodies are described in US 2011/0229489 and 2014/0044730, which are incorporated by reference herein.

In one embodiment the anti-PCSK9 portion provides sustained suppression of free PCSK9. In one embodiment, is provides at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% suppression.

In one embodiment, an anti-PCSK9 antibody or antigen binding fragment thereof capable of specifically binding PCSK9 comprises:

-   -   a. a heavy chain variable region comprising a sequence which is         at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% A         identical to SEQ ID NO: 1, 5, 8, or 10; and     -   b. a light chain variable region comprising a sequence which is         at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% A         identical to SEQ ID NO: 2, 6, 9 or 11.

In one embodiment, an anti-PCSK9 antibody or antigen binding fragment thereof capable of specifically binding PCSK9 comprises:

-   -   a. a heavy chain variable region CDRI sequence comprising a         sequence which has one mutation compared to SEQ ID NO: 14;     -   b. a heavy chain variable region CDR2 sequence comprising a         sequence which has one or two mutations compared to SEQ ID NO:         15, 16, 17, or 18;     -   c. a heavy chain variable region CDR3 sequence comprising a         sequence which is one mutation compared to SEQ ID NO: 19.     -   d. a light chain variable region CDRI sequence comprising a         sequence which has one or two mutations compared to SEQ ID NO:         20, 21, 22, or 23;     -   e. a light chain variable region CDR2 sequence comprising a         sequence which has one mutation compared to SEQ ID NO: 24 or 25;         and     -   f. a light chain variable region CDR3 sequence comprising a         sequence which has one mutation compared to SEQ ID NO: 26.

In one embodiment, an anti-PCSK9 antibody or antigen binding fragment thereof capable of specifically binding PCSK9 comprises:

-   -   a. a heavy chain variable region CDRI sequence comprising SEQ ID         NO: 14;     -   b. a heavy chain variable region CDR2 sequence comprising SEQ ID         NO: 15, 16, 17, or 18;     -   c. a heavy chain variable region CDR3 sequence comprising SEQ ID         NO: 19.     -   d. a light chain variable region CDRI sequence comprising SEQ ID         NO: 20, 21, 22, or 23;     -   e. a light chain variable region CDR2 sequence comprising SEQ ID         NO: 24 or 25; and     -   f. a light chain variable region CDR3 sequence comprising SEQ ID         NO: 26.

In another embodiment, the anti-PCSK9 portion may comprise an anti-PCSK9 antibody or antigen-binding fragment as described in any of U.S. Pat. No. 8,030,457, U.S. Pat. No. 8,062,640, U.S. Pat. No. 8,357,371, U.S. Pat. No. 8,168,762, U.S. Pat. No. 8,563,698, U.S. Pat. No. 8,829,165, U.S. Pat. No. 8,859,741, U.S. Pat. No. 8,188,233, WO 2012/088313, US 2012/0195910, U.S. Pat. No. 8,530,414, US 2013/0189278, U.S. Pat. No. 8,344,144, US 2011/0033465, U.S. Pat. No. 8,188,234, U.S. Pat. No. 8,080,243, US 2011/0229489, US 2010/0233177, US 2013/315927 and US 2013/0071405. Each of these references is incorporated reference for the sequence and description of anti-PSCK9 antibodies and antigen-binding fragments thereof.

In one embodiment, the anti-PCSK9 portion may comprise a heavy and light chain variable region chosen from the sequences in Group B of Table 1 above. Alternatively, the anti-PCSK9 portion may comprise a heavy and light chain variable region with CDRs identical to a heavy and light chain variable region from Group B of Table 1 above. Additionally, the anti-PCSK9 portion may comprise a heavy chain variable region chosen from SEQ ID NOS: 53, 55, 57, 59, 61, or 63 and a light chain variable region chosen from SEQ ID NOS: 54, 56, 58, 60, 62, or 64, Alternatively, the anti-PCSK9 portion may comprise heavy chain CDR1, CDR2, and CDR3 from any one of SEQ ID NOS: 53, 55, 57, 59, 61, or 63 and light chain CDR1, CDR2, and CDR3 from any one of SEQ ID NOS: 54, 56, 58, 60, 62, or 64.

In one embodiment, the anti-PCSK9 portion may comprise a heavy and light chain variable region chosen from the sequences in Group C of Table 1 above. Alternatively, the anti-PCSK9 portion may comprise a heavy and light chain variable region with CDRs identical to a heavy and light chain variable region from Group C of Table 1 above. Additionally, the anti-PCSK9 portion may comprise a heavy chain variable region chosen from SE(ID NOS: 65-95 and a light chain variable region chosen from SEQ ID NOS: 96-126. Alternatively, the anti-PCSK9 portion may comprise heavy chain CDR1, CDR2, and CDR3 from any one of SEQ ID NOS: 65-95 and light chain CDR1 CDR2, and CDR3 from any one of SEQ ID NOS: 96-126.

In one embodiment, the anti-PCSK9 portion may comprise a heavy and light chain variable region chosen from the sequences in Group D of Table 1 above. Alternatively, the anti-PCSK9 portion may comprise a heavy and light chain variable region with CDRs identical to a heavy and light chain variable region from Group D of Table 1 above. Additionally, the anti-PCSK9 portion may comprise a heavy chain variable region chosen from SECS ID NOS: 127-218 and a light chain variable region chosen from SEQ ID NOS: 219-311. Alternatively, the anti-PCSK9 portion may comprise heavy chain CDR1, CDR2, and CDR3 from any one of SEQ ID NOS: 127-218 and light chain CDR1 CDR2, and CDR3 from any one of SEQ ID NOS: 219-311.

In one embodiment, the anti-PCSK9 portion may comprise a heavy and light chain variable region chosen from the sequences in Group E of Table 1 above. Alternatively, the anti-PCSK9 portion may comprise a heavy and light chain variable region with CDRs identical to a heavy and light chain variable region from Group E of Table 1 above. Additionally, the anti-PCSK9 portion may comprise a heavy chain variable region chosen from SEQ ID NOS: 312-317 and a light chain variable region chosen from SEQ ID NOS: 318-323. Alternatively, the anti-PCSK9 portion may comprise heavy chain CDR1, CDR2, and CDR3 from any one of SEQ ID NOS: 312-317 and light chain CDR1, CDR2, and CDR3 from any one of SEQ ID NOS: 318-323.

In one embodiment, the anti-PCSK9 portion may comprise a heavy and light chain variable region chosen from the sequences in Group F of Table 1 above. Alternatively, the anti-PCSK9 portion may comprise a heavy and light chain variable region with CDRs identical to a heavy and light chain variable region from Group F of Table 1 above. Additionally, the anti-PCSK9 portion may comprise a heavy chain variable region chosen from SECS ID NO: 324 and a light chain variable region chosen from SEQ ID NO: 325. Alternatively, the anti-PCSK9 portion may comprise heavy chain CDR1, CDR2, and CDR3 from any one of SEQ ID NOS: 324 and light chain CDR1, CDR2, and CDR3 from any one of SEQ ID NOS: 325.

In one embodiment, the anti-PCSK9 portion may comprise a heavy and light chain variable region chosen from the sequences in Group G of Table 1 above. Alternatively, the anti-PCSK9 portion may comprise a heavy and light chain variable region with CDRs identical to a heavy and light chain variable region from Group G of Table 1 above. Additionally, the anti-PCSK9 portion may comprise a heavy chain variable region chosen from SEQ ID NOS: 326, 339 340, or 343-400 and a light chain variable region chosen from SEQ ID NOS: 327, 341, or 342. Alternatively, the anti-PCSK9 portion may comprise heavy chain CDR1, CDR2, and CDR3 from any one of SEQ ID NOS: 326, 339, 340, or 343-400 and light chain CDR1, CDR2, and CDR3 from any one of SEQ ID NOS: 327, 341, or 342. Further, the anti-PCSK9 portion may comprise heavy chain CDR1, CDR2, and CDR3 from SEQ ID NOS: 328 (HC CDR1) 329 (HC CDR2), 331 (HC CDR2), 330 (HC CDR3), 332 (HC CDR3) and light chain CDR1 CDR2, and CDR3 from SEQ ID NOS: 333 (LC CDR1), 334 (LC CDR2), 333 (LC CDR3), 336 (LC CDR1), (LC CDR2), and 338 (LC CDR3). In one embodiment the anti-PCSK9 portion may comprise a heavy variable region comprising the amino acid sequence of SEQ ID NO: 491 and light chain variable region comprising the amino acid sequence of SEQ ID NO: 492. Alternatively, the anti-PCSK9 portion may comprise heavy chain CDR1, CDR2, and CDR3 from SEQ ID NOS: 493-495 and light chain CDR1, CDR2, and CDR3 from SEQ ID NOS: 496-498.

In other embodiments, the antibody portion may comprise antibodies other than an anti-PCSK9 antibody (e.g., an anti-B7-H1 antibody). In one embodiment, the anti-B7-H1 antibody may comprise a heavy variable region comprising the amino acid sequence of SEQ ID NO: 422 and light chain variable region comprising the amino acid sequence of SEQ ID NO: 423.

2. Antibody or Antigen-Binding Fragments

As used herein, the term antibody or antigen-binding fragment thereof is used in the broadest sense. It may be man-made such as monoclonal antibodies (mAbs) produced by conventional hybridoma technology, recombinant technology and/or a functional fragment thereof. It may include both intact immunoglobulin molecules for example a polyclonal antibody, a monoclonal antibody (mAb), a monospecific antibody, a bispecific antibody, a polyspecific antibody, a human antibody, a humanized antibody, an animal antibody (e.g. camelid antibody), chimeric antibodies, as well as portions, fragments, regions, peptides and derivatives thereof (provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis, or recombinant techniques), such as, for example, immunoglobulin devoid of light chains, Fab, Fab′, F (ab′)₂, Fv, scFv, antibody fragment, diabody, Fd, CDR regions, or any portion or peptide sequence of the antibody that is capable of binding antigen or epitope. In one embodiment, the functional part is a single chain antibody, a single chain variable fragment (scFv), a Fab fragment, or a F(ab′)₂, fragment.

An antibody or functional part is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. Antibody fragments or portions may lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. Examples of antibody may be produced from intact antibodies using methods well known in the art, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)₂, fragments). Portions of antibodies may be made by any of the above methods, or may be made by expressing a portion of the recombinant molecule. For example, the CDR region(s) of a recombinant antibody may be isolated and subcloned into an appropriate expression vector.

In one embodiment, an antibody or functional part is a human antibody. The use of human antibodies for human therapy may diminish the chance of side effects due to an immunological reaction in a human individual against nonhuman sequences. In another embodiment, the antibody or functional part is humanized. In another embodiment, an antibody or functional part is a chimeric antibody. This way, sequences of interest, such as for instance a binding site of interest, can be included into an antibody or functional part.

In one embodiment, the antibody may have an IgG, IgA, IgM, or IgE isotype. In one embodiment, the antibody is an IgG. In one aspect, the anti-PCSK9 antibody or antigen-binding fragment thereof may be an IgG 1.

3. Modifications to the Constant Domain

In one embodiment, the anti-PCSK9 antibody or antigen binding fragment comprises an Fc region. It will be understood that Fc region as used herein includes the polypeptides comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). The “EU index as set forth in Kabat” refers to the residue numbering of the human IgG1 EU antibody as described in Kabat et al. supra. Fc may refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein.

In one embodiment, the anti-PCSK9 antibody or antigen-binding portion thereof has a variant Fc region having reduced effector function (e.g., reduced ADCC and/or CDC). In one embodiment, the Fc region has no detectable effector function. In one embodiment, the Fc region comprises at least one non-native amino acid at one or more positions chosen from 234, 235, and 331, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the present invention provides an Fc variant, wherein the Fc region comprises at least one non-native amino acid chosen from 234F, 235F, 235Y, and 331S, as numbered by the EU index as set forth in Kabat. In a further specific embodiment, an Fc variant of the invention comprises the 234F, 235F, and 331S amino acid residues, as numbered by the EU index as set forth in Kabat. In another specific embodiment, an Fc variant of the invention comprises the 234F, 235Y, and 331S amino acid residues, as numbered by the EU index as set forth in Kabat. In a particular embodiment, the anti-PCSK9 antibody or antigen-binding portion thereof has a variant Fc region, wherein the variant comprises a phenylalanine (F) residue at position 234, a phenylalanine (F) residue or a glutamic acid (E) residue at position 235 and a serine (S) residue at position 331, as numbered by the EU index as set forth in Kabat. Such mutation combinations are hereinafter referred to as the triple mutant (TM).

The serine228proline mutation (S228P), as numbered by the EU index as set forth in Kabat, hereinafter referred to as the P mutation, has been reported to increase the stability of a particular IgG4 molecule (Lu et al., J Pharmaceutical Sciences 97(2):960-969, 2008). Note: In Lu et al. it is referred to as position 241 because therein they use the Kabat numbering system, not the “EU index” as set forth in Kabat.

This P mutation may be combined with L235E to further knock out ADCC. This combination of mutations is hereinafter referred to as the double mutation (DM).

B. GLP-1 Moiety

The fusion molecule contains a GLP-1 moiety. GLP-1 may also be referenced by the synonym glucagon-like peptide-1. By GLP-1 we also reference Exendin-4, which is a GLP-1 analog. In one embodiment, the GLP-1 moiety provides glucose control and/or weight loss benefits.

In one embodiment, the full length GLP-1 molecule may be used in the fusion protein. In another embodiment, a fragment of GLP-1 may be used as a GLP-1 moiety in the fusion protein.

In one embodiment, the GLP-1 moiety has a pair of cysteine residues that allows for a disulphide bridge. In one embodiment, the cysteine is an engineered cysteine compared to the parental sequence. In one embodiment, the cysteine is an E18C mutation.

In one embodiment, the GLP-1 potency is reduced at the human GLP-1 receptor compared to wild type GLP-1 (e.g., SEQ ID NO: 29) or a GLP-1 analog (e.g., SEQ ID NO: 12). In another embodiment, the GLP-1 potency at the human GLP-1 receptor is at least about 10×, 20×, 30×, 40×, 50×, 60×, 100×, 125×, 150×, 175×, 200×, or 225× lower than wildtype GLP-1 or a GLP-1 analog. In another embodiment, the potency is reduced by similar amounts as compared to dulaglutide. The GLP-1 potency may be reduced to allow for saturation of PCSK9 while reducing side effects. In one embodiment, the GLP-1 moiety may have at least one mutation that reduces the potency of the GLP-1 moiety. In one embodiment, this offers benefits of reducing side effects, including, but not limited to nausea. In one embodiment, the mutation is a point mutation. In one embodiment, the point mutation is chosen from V19A, G2V, E15A, or L26I with respect to Exendin-4.

In one embodiment, the GLP-1 moiety is comprises any one of SEQ ID NOS: 3, 7, 12, 13, or 28-42. In another embodiment, the GLP-1 moiety is a fragment of any one of SEQ ID NOS: 3, 7, 12, 13, or 28-42 comprising at least 10, 15, 20, or 25 amino acids. In a further embodiment, the GLP-1 moiety comprises a sequence which is at least at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 3, 7, 12, 13, or 28-42. In a further embodiment, the GLP-1 moiety comprises a sequence which has 1, 2, 3, 4, 5, or 6 mutations as compared to any one of SEQ ID NOS: 3, 7, 12, 13, or 28-42.

C. Fusion and Linkers

In one embodiment, the GLP-1 moiety is fused directly or indirectly to the light chain of the anti-PCSK9 antibody or antigen binding fragment. In another embodiment, the GLP-1 moiety is fused directly or indirectly to the heavy chain of the anti-PCSK9 antibody or antigen binding fragment.

In one embodiment, a linker may be used to construct a fusion between the anti-PCSK9 antibody or antigen-binding fragment and the GLP-1 moiety. In another embodiment, the anti-PCSK9 antibody or antigen-binding fragment may be directly conjugated to the GLP-1 moiety.

If a linker is used, the linker may be chosen from any suitable linker for fusion proteins. In one embodiment, the linker may comprise a GGGGS (SEQ ID NO: 27) repeat, either alone or in combination with other amino acids, either as one, two, three, or four sets of repeats. In one embodiment, the linker may comprise other combinations of G and S, either alone or in combination with other amino acids. In some embodiments, the linker has a C-terminal Alanine (A).

In one embodiment, a specific linker may be chosen from GGGGSGGGGSGGGGSA (SEQ ID NO: 4). In one embodiment, the linker allows for a disulfide bridge to form between the C terminus of the GLP-1 moiety and another portion of the GLP-1 molecule, but not with the antibody portion. In such an instance, a linker with limited flexibility may prevent undesired disulfide bridging to the antibody portion.

In one embodiment, the linker is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4.

In one embodiment, the cysteine:cysteine disulfide bridge is formed by making a cysteine substitution mutation in the GLP-1 moiety. In one embodiment, the mutation is E18C.

II. Nucleic Acids Encoding Fusion Molecules

The present embodiments further provides an isolated, synthetic, or recombinant nucleic acid sequence encoding any of the fusion molecules described in section I above. Such nucleic acids encode the heavy and light chain sequences set forth herein. Alternatively, such nucleic acids include the anti-PCSK9 antibody or antigen-binding portion fused to the GLP-1 moiety portion. Due to the degeneracy of the nucleic acid code, multiple nucleic acids will encode the same amino acid and all are encompassed herein.

III. Methods of Making Fusion Molecules, Formulation, and Pharmaceutical Compositions

One embodiment includes a method of producing the fusion molecule by culturing host cells under conditions wherein a nucleic acid is expressed to produce the fusion molecule, followed by recovering the fusion molecule. A variety of cell lines may be used for expressing the fusion molecule, including, but not limited to, mammalian cell lines. In one embodiment, the cell lines may be human. In another embodiment, bacterial or insect cell lines may be used. In one embodiment, the cell lines include Chinese hamster ovary (CHO) cells, variants of CHO cells (for example DG44), 293 cells, and NSO cells. In another embodiment, cell lines include VERY, BHK, Hela, COS, MDCK, 293F, 293T, 3T3, W138, BT483, Hs578T, Sp2/0, HTB2, BT2O, T47D, CRL7030, and HsS78Bst cells.

Recombinant expression utilizes construction of an expression vector containing a polynucleotide that encodes the fusion molecule. Once a polynucleotide has been obtained, a vector for the production of the fusion molecule may be produced by recombinant DNA technology well known in the art. Expression vectors may include appropriate transcriptional and translational control signals. This may be accomplished using in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. In one embodiment, a replicable vector comprises a nucleic acid sequence encoding an antibody or functional part operably linked to a heterologous promoter.

A variety of host-expression vector systems may be utilized to express the fusion molecule as described in U.S. Pat. No. 5,807,715. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus, are an effective expression system for antibodies (Foecking et al., Gene, 45:101 (1986); and Cockett et al., Bio/Technology, 8:2 (1990)). In addition, a host cell strain may be chosen which modulates the expression of inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the protein of the invention. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.

In bacterial systems, a number of expression vectors may be selected depending upon the use intended for the fusion molecule being expressed. For example, when a large quantity of such fusion molecule is to be produced, for the generation of pharmaceutical compositions comprising the fusion molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO, 12:1791 (1983)), in which the coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, 1989, J. Biol. Chem., 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione-S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to glutathione-agarose affinity matrix followed by elution in the presence of free glutathione. The pGEX vectors are designed to introduce a thrombin and/or factor Xa protease cleavage sites into the expressed polypeptide so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The protein coding sequence may be cloned individually into non-essential regions (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedrin promoter).

In mammalian host cells, a number of virus based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody or functional part in infected hosts (e.g., see, Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody or functional part coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon should generally be in frame with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., Methods in Enzymol., 153:51-544(1987)).

Stable expression can be used for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express the fusion molecule may be generated. Host cells can be transformed with an appropriately engineered vector comprising expression control elements (e.g., promoter, enhancer, transcription terminators, polyadenylation sites, etc.), and a selectable marker gene. Following the introduction of the foreign DNA, cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells that stably integrated the plasmid into their chromosomes to grow and form foci which in turn can be cloned and expanded into cell lines. Plasmids that encode the fusion molecule can be used to introduce the gene/cDNA into any cell line suitable for production in culture.

A number of selection systems may be used, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell, 11:223 (1977)), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell, 22:8-17 (1980)) genes can be employed in tk-, hgprt- or aprT-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA, 77:357 (1980); O′Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIB TECH 11(5):155-2 15 (1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene, 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol., 150:1.

Once the fusion molecule has been produced by recombinant expression, it may be purified by any method known in the art for purification, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigens Protein A or Protein G, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

Further provided are compositions, e.g., pharmaceutical compositions, that contain an effective amount of a dual active fusion molecule as provided herein, formulated for the treatment of metabolic diseases, e.g., Type 2 diabetes.

Compositions of the disclosure can be formulated according to known methods. Suitable preparation methods are described, for example, in Remington's Pharmaceutical Sciences, 19th Edition, A. R. Gennaro, ed., Mack Publishing Co., Easton, Pa. (1995), which is incorporated herein by reference in its entirety. Composition can be in a variety of forms, including, but not limited to an aqueous solution, an emulsion, a gel, a suspension, lyophilized form, or any other form known in the art. In addition, the composition can contain pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives. Once formulated, compositions of the disclosure can be administered directly to the subject.

Carriers that can be used with compositions of the disclosure are well known in the art, and include, without limitation, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, and polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. Compositions can be sterilized by conventional, well known sterilization techniques, or can be sterile filtered. A resulting composition can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. Compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

IV. Methods for Use of the Anti-PCSK9˜GLP-1 Fusion Molecule and Kits

The anti-PCSK9˜GLP-1 fusion may be used to treat diabetes or TType 2 diabetes. In one embodiment, the patient has TType 2 diabetes. In one embodiment, the patient has a high cardiovascular risk profile. In another embodiment, the patient has both TType 2 diabetes and a high cardiovascular risk profile. By high cardiovascular risk profile, it means that the patient is at higher risk for a cardiovascular event due to one or more factors: high cholesterol, high LDL cholesterol, low HDL cholesterol, high blood pressure, atherosclerosis, obesity, prior cardiovascular event (including angina, heart attack, transient ischemic attack, stroke, etc.), family history of cardiovascular event, smoking, high triglycerides, lack of physical activity, poorly controlled blood sugars, and the like.

In other embodiments, the anti-PCSK9˜GLP-1 fusion may be used to treat other disease including but not limited to NASH, obesity, hypercholesterolemia, and major adverse cardiovascular events (MACE) including but not limited to acute coronary syndrome (ACS), stroke, heart failure, and malignant dysrhythmia.

In one embodiment, the fusion molecule has increased stability upon administration. In one embodiment, the increased stability is demonstrated by comparing it to a benchmark control compound dulaglutide, a GLP-1 analog fused with an Fc fragment, in an in vivo administration to mice.

In one embodiment, the fusion molecule as increased potency at the GLP-1 receptor. In one embodiment, the decreased potency is demonstrated at the human GLP-1 receptor over the benchmark control compound dulaglutide and/or wild type GLP-1.

In some embodiments, the fusion molecule promotes weight loss in a subject.

In one embodiment, the fusion molecule is administered by injection.

In other embodiments, the present disclosure provides kits comprising dual active fusion molecules, which can be used to perform the methods described herein. In certain aspects, a kit comprises a dual active fusion molecule disclosed herein in one or more containers. A kit as provided herein can contain additional compositions for combination therapies. One skilled in the art will readily recognize that the disclosed dual active fusion molecules can be readily incorporated into one of the established kit formats that are well known in the art.

Reference will now be made in detail to the present exemplary embodiments, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice disclosed herein. The embodiments are further explained in the following examples. These examples do not limit the scope of the claims, but merely serve to clarify certain embodiments. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit being indicated by the following claims.

EXAMPLES Example 1 Antibody Optimisation

The anti-PCSK9 antibody PC9#2 (SEQ ID NOs. 8 and 9 for the variable heavy and light chains respectively) has been optimised to:

1_ Reduce the immunogenicity risk by reverting amino acids to the ones corresponding to the closest human germline sequence without significantly impacting the binding to PCSK9 antigen.

2_ Remove pH-dependent binding to PCSK9 by mutating histidine residues VH_52, VL_30 and VL_50 (Kabat numbering).

3_ Improve affinity for human PCSK9 at physiological pH in order to efficiently engage with the target and achieve sufficient free PCSK9 suppression following administration of PCSK9/GLP-1 peptide antibody fusion molecules.

A) Germlining

The amino acid sequences of the VH and VL domains of the anti-PCSK9 antibody PC9#2 were aligned to the known human germline sequences in the VBASE database (Tomlinson, 1997; http://vbase.mrc-cpe.cam.ac.uk/), and the closest human germline was identified by sequence similarity to be 1-46 (DP-7) (SEQ ID 417) and VK1 O18 O8 (DPK1) (SEQ ID 418) for the variable heavy and light chains respectively. FIGS. 1E and 1F are showing an alignment of PC9#2 variable domains with those germline sequences.

A structure model of the anti-PCSK9 PC9#2 antibody in complex with human PCSK9 antigen has been generated using the primary amino acid sequence of PC9#2 variable domains and previously described crystal structure of human PCSK9 in complex with another anti-PCSK9 antibody deposited in the Protein Data Bank using PBD ID code 3SQO (Liang et al., 2012, J. Pharm. Exp. Ther., Vol. 340, p228-236).

Using that structure model, the following residues were identified as non-contacting with PCSK9 antigen but nonetheless solvent-exposed: Arg56, Asn58, Glu61, Lys64 and Ser65 in the variable heavy chain as well as Arg24 in the variable light chain (Kabat numbering). Because those residues are different from the closest germline sequences and might be solvent-exposed, they can present some immunogenicity risks. Without being bound by theory, mutating those residues should not significantly impact the ability of the antibody to strongly bind PCSK9 as they should not contribute to the interaction network between the antibody and its antigen.

Mutations R56S, N58S, E61Q, K64Q and S65G were then introduced in PC9#2 heavy chain sequence as well as K24Q in the light chain using standard molecular biology techniques to generate the antibody PC9#2_FG of SEQ ID 5 and 6 for the variable heavy and light chains respectively.

Anti-PCSK9 PC9#2 and PC9#2_FG were produced as human IgG1-TM antibodies as described in Example 2 and characterised for binding to human PCSK9 using Biacore as described in Example 17. Kinetic parameters at pH 7.4 for those compounds are summarised in Table 2. Both antibodies exhibit similar on-rate, off-rate and affinity for human PCSK9 demonstrating that the germlining mutagenesis had no impact on antigen binding.

TABLE 2 Kinetic parameters for human PCSK9 at physiological pH of germlined and non-germlined version of PC9#2antibody Compound ka (1/Ms) kd (1/s) KD (M) PC9#2 2.70E+05 5.10E−04 1.89E−09 PC9#2_FG 3.03E+05 5.40E−04 1.78E−09

B) Removing pH Dependent Binding and Improving Affinity For Human PCSK9

Anti-PCSK9 antibodies PC9#2 and PC9#2_FG exhibit pH-dependent binding properties as they bind strongly to human PCSK9 antigen at physiological pH but rapidly dissociate from their target at acidic pH. That feature should enable the antibodies to dissociate from PCSK9 in the acidic compartment of endosome in order to be recycled at cell surface rather than being sent to lysosome for degradation. This should ultimately translate into a longer antibody in vivo half-life.

Table 3 are comparing off-rate (kd) for those two compound with human PCSK9 at pH7.4 (physiological) and 6.0 (acidic) determined by Biacore as described in Example 17 but with the following modifications. After antibody capture onto the CM5 chip, human PCSK9 antigen diluted to concentrations ranging from 1 nM to 200 nM in running buffer pH7.4 (10 mM sodium phosphate pH 7.4, 150 mM sodium chloride, 1 mg/mL BSA, 0.05% Tween20) were injected for 10 minutes. After the association phase, running buffer at pH7.4 or pH6.0 were injected for 10 minutes dissociation phase. Global dissociation rates were calculated using a 1:1 binding kinetics model.

TABLE 3 Dissociation constant (kd) at physiological and acidic pH of germlined and non-germlined version of PC9#2antibody. kd (1/s) kd (1/s) Compound pH 7.4 pH 6.0 kd ratio PC9#2 5.10E−04 3.24E−03 6.4 PC9#2_FG 5.40E−04 6.47E−03 12

Long antibody in vivo half-life is not desirable for PCSK9/GLP-1 fusion molecules as it might lead to the accumulation of drug metabolites able to bind PCSK9 but degraded in the GLP-1 analogue peptide and thus unable to activate the GLP-1 receptor.

In addition, genetic fusion of a GLP-1 analogue peptide in front of the light chain of the anti-PCSK9 PC9#2 was slightly impacting the affinity of the compound for human PCSK9 (19 nM) compared to PC9#2 antibody (7 nM) as shown in Example 5, Table 11. Affinity maturation of PC9#2_FG was then required in order to counterbalance the negative impact of GLP-1 analogue light chain fusion on affinity for human PCSK9 antigen.

To remove pH-dependent binding, histidine residues in position 52 of the heavy chain as well as in position 30 and 50 of the light chain need to be mutated. Based on the structure model described above and subsequent analysis of PC9#2 in complex with human PCSK9, the following mutations were identified as potentially beneficial for antibody binding to PCSK9 and could lead to an affinity improvement: heavy chain H52K or H52S, light chain H30Y, H30K or H30S and light chain H50Y or H50S.

Combination of all those mutations in the sequence of PC9#2_FG were generated using standard molecular biology techniques in order to produce optimised antibodies. Table 4 is summarising the different compounds resulting from the combination experiment.

TABLE 4 PC9#2_FG mutations to generate optimised antibodies Antibody # VH_H52 VL_H30 VL_H50 name SEQ ID 1 K Y Y HS1 424 and 425 2 K Y S HS2 426 and 427 3 K K Y HS3 428 and 429 4 K K S HS4 430 and 431 5 K S Y / 6 K S S HS5 432 and 433 7 S Y Y HS6 434 and 435 8 S Y S HS7 436 and 437 9 S K Y HS8 438 and 439 10 S K S HS9 1 and 2 11 S S Y / 12 S S S HS10 442 and 443

Combination of H30S and HSOY mutations in PC9#2_FG light chain failed to deliver and compound #5 and #11 have subsequently not been generated.

Antibodies were produced as human IgG1-TM as described in Example 2 and tested for their ability to block the binding of PC9#2 to human PCSK9 using an epitope competition assay as described in Example 3. Data are summarised in Table 5 and show that HS7, HS9 and HS10 have an IC50 at least 10-fold lower compared to the parent antibody PC9#2_FG suggesting that those antibodies may have a significantly better affinity for PCSK9 than PC9#2_FG.

TABLE 5 Inhibition of PC9#2 binding to human PCSK9 using anti-PCSK9 antibodies as competition reagent. Ratio over Antibody IC50 (M) PC9#2_FG HS1 5.6E−10 1.3 HS2 9.3E−11 7.8 HS3 7.0E−10 1.0 HS4 1.2E−10 6.1 HS5 2.2E−10 3.3 HS6 1.7E−10 4.3 HS7 6.3E−11 11.6 HS8 1.6E−10 4.6 HS9 6.6E−11 11.1 HS10 7.0E−11 10.4 PC9#2_FG 7.3E−10 1.0

Antibody HS9 has been further characterised and compared to the parental antibody PC9#2_FG for its binding parameters to human PCSK9 at physiological pH by Biacore as described in Example 17. As shown in Table 6, engineered anti-PCSK9 antibody HS9 displays a 3-fold improvement in affinity for human PCSK9 compared to PC9#2_FG at physiological pH, mainly due to a reduction in off-rate (kd).

TABLE 6 Kinetic parameters for human PCSK9 at physiological pH of engineered antibody HS9 compared to PC9#2_FG Antibody ka (1/Ms) kd (1/s) KD (M) PC9#2_FG 3.09E+05 6.57E−04 2.13E−09 HS9 3.81E+05 2.57E−04 6.75E−10

In a separate experiment, pH dependent binding of HS9 anti-PCSK9 antibody has been compared to PC9#2_FG using Biacore as described above. Table 7 is showing the dissociation constant (kd) at physiological and acidic pH for those two compounds. Contrary to PC9#2_FG which dissociates more rapidly at pH6.0 than at pH7.4, HS9 displays a lower kd at acidic than at physiological pH demonstrating that it is dissociating from PCSK9 more slowly at pH6.0 than at pH7.4.

TABLE 7 Dissociation constant (kd) at physiological and acidic pH of engineered antibody HS9 compared to PC9#2_FG. kd (1/s) kd (1/s) Antibody pH 7.4 pH 6.0 ratio kd PC9#2_FG 6.50E−04 7.40E−03 11.4 HS9 2.04E−04 9.82E−05 0.5

Example 2 Preparation of a Dual Action Fusion Molecule

A dual action fusion molecule was made according with a large-scale structure as shown in FIG. 1A of: GLP-1 moiety—Linker—Antibody Light Chain. SEQ ID NO: 3 was used as the GLP-1 moiety, SEQ ID NO: 4 was used as the linker, and SEQ ID NO: 1 and 2, were used as the heavy and light chain respectively.

In this embodiment, the N-terminal end of GLP-1 analogue peptides was free in order to most efficiently engage and activate the GLP-1 receptor; peptides were fused to the N-terminal of antibody variable domains. A linker sequence was used in many constructs between the end of the peptide and the start of variable domain in order to minimize the impact of the fusion on peptide and/or antibody activities. Peptides were fused either at the heavy chain or the light chain of antibodies in order to obtain two peptide moieties per fusion molecule. Large scale structure of such fusions are shown FIG. 1B (heavy chain fusion) and 1C (light chain fusion). In some embodiments, peptides may be fused at both the heavy and light chains to display four peptide moieties per fusion molecule (shown prophetically in FIG. 1D).

Genes coding for GLP-1 analogue peptides in fusion with antibody variable domains were built by overlapping PCR using standard methods. Unique restriction sites were incorporated at the 5′ and 3′ end of the DNA fragment to enable cloning in the expression vectors.

The VH domain, with or without a peptide/linker fusion, was cloned into a vector containing the human heavy chain constant domains and regulatory elements to express whole IgG1 triple mutant (IgG1-TM) heavy chain in mammalian cells. IgG1-TM format is similar to human IgG1 but Fc sequence incorporating mutations L234F, L235E and P331S to reduce its ability to trigger antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity (Oganesyan V. et al., 2008, Acta Cryst., D64: 700-704). The VL domain, with or without peptide/linker fusion, was cloned into a vector for the expression of the human light chain constant domains and regulatory elements to express whole IgG light chain in mammalian cells. An OriP fragment was included in the heavy and light chain expression vectors to facilitate use with CHO cells and to allow episomal replication.

To obtain IgGs and peptide antibody fusions, the heavy and light chain expressing vectors were transiently transfected into 30 mL (small scale) or 400 mL (medium scale) of CHO mammalian cells. Table 8 is summarizing the different products that can be obtained by co-transfection of the heavy and light chain expressing vectors with or without a peptide/linker fusion.

TABLE 8 Product description when using heavy and light chain vectors co-transfection # Heavy chain vector Light chain vector Product 1 VH without peptide VL without peptide IgG fusion fusion 2 VH with peptide VL without peptide Peptide/antibody VH fusion fusion fusion (FIG. 1B) 3 VH without peptide VL with peptide Peptide/antibody VL fusion fusion fusion (FIG. 1C) 4 VH with peptide VL with peptide Peptide/antibody fusion fusion VH + VL (shown prophetically in FIG. 1D)

Compounds were expressed and secreted into the medium. Harvests were pooled and filtered before compound purification using Protein A chromatography. Culture supernatants were loaded on a column of appropriate size of MabSelectSure (GE Healthcare Life Sciences) and washed with 1×DPBS (Gibco). Bound compound was eluted from the column using 0.1 M Sodium Citrate pH 3.0 and neutralised by the addition of Tris-HCl pH 9.0. For small scale transfection, eluted material was buffer exchanged into 1×DPBS using PD10 columns (GE Healthcare). For medium scale transfection, eluted material was further purified by Size Exclusion Chromatography (SEC) using either a HiLoad 16/600 Superdex 200 prep grade column (GE Healthcare) for sample volumes of up to 5 ml or a HiLoad 26/600 Superdex 200 column (GE Healthcare) for sample volumes of up to 12 ml. Isocratic elution was performed using 1×DPBS as the running buffer.

Compound concentration was determined spectrophotometrically using an extinction coefficient based on the amino acid sequence according to the protocol in Mach, H. et al., Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins, Anal Biochem, 200(1):74-80 (1992). Purified compounds were analysed for aggregation and degradation using SEC-HPLC and SDS-PAGE. SEC-HPLC was performed by loading 70 μL of sample onto a TSKgel G3000SWXL 7.8 mm×300 mm column (Tosoh Bioscience) using a flow rate of 1 mL/min and 0.1M Sodium Phosphate Dibasic anhydrous plus 0.1M Sodium Sulphate at pH 6.8 as running buffer. SDS-PAGE is run by loading 2 μg protein on a Nu PAGE® 4%-12% Bis-Tris (Invitrogen) using 1× Nu PAGE® MES SDS Running Buffer (Invitrogen). Compounds from medium scale transfections were further characterized for integrity by Electro-Spray Ionisation Mass Spectrometry (ESI-MS) and tested for endotoxin level using the Limulus Amebocyte Lysate (LAL) Kinetic-QCL (Lonza). For ESI-MS analysis, samples were prepared at 1 mg/mL in 10 mM Tris-HCl pH 8.0. A 30 minutes reduction at 37° C. using 10 mM DTT was carried out prior analysis. Data were acquired using a Waters ACQUITY UPLC® I-Class system coupled to a Waters SYNAPT G1 QTOF Mass Spectrometer, operated using MassLynx software. The mobile phases used were highly purified water plus 0.01% trifluoroacidic acid (TFA), 0.1% formic acid (A) and acetonitrile +0.01% TFA, 0.1% FA (B). Separation between heavy and light chain was achieved using a 2.1 mm×50 mm Waters BEH300 C4 column, heated at 60° C. The flow rate was set at 0.3 mL/min, with 22 minutes total run time. Samples (10 pmol) were injected onto the column and MS data were acquired in positive ion mode, using the following electrospray source parameters: capillary voltage: 3.4 kV, 81° C. source temperature: 81° C., desolvation temperature: 24° C. Data were acquired between 500 and 4500 Da.

Example 3 In Vitro Characterization of a GLP-1 Analogue in Genetic Fusion with Anti-PCSK9 Antibodies

To evaluate the feasibility of generating dual activity molecules by genetic fusion between GLP-1 receptor agonist peptides and anti-PCSK9 antibodies, the GLP-1 analogue peptide of SEQ ID NO:28 was fused using a linker of SEQ ID NO:4 to the heavy or light chain variable domains of seven different previously identified anti-PCSK9 antibodies.

1_ PC9#1 with antibody variable heavy chain of SEQ ID NO:10 and antibody variable light chain of SEQ ID NO:11.

2_ PC9#2 with antibody variable heavy chain of SEQ ID NO:8 and antibody variable light chain of SEQ ID NO:9.

3_ PC9#3 with antibody variable heavy chain of SEQ ID NO: 404 and antibody variable light chain of SEQ ID NO: 405.

4_ PC9#4 with antibody variable heavy chain of SEQ ID NO: 406 and antibody variable light chain of SEQ ID NO: 407.

5_ PC9#5 with antibody variable heavy chain of SEQ ID NO: 408 and antibody variable light chain of SEQ ID NO: 409.

6_ PC9#6 with antibody variable heavy chain of SEQ ID NO: 410 and antibody variable light chain of SEQ ID NO: 411.

7_ PC9#7 with antibody variable heavy chain of SEQ ID NO: 412 and antibody variable light chain of SEQ ID NO:413.

PCSK9 activity of each peptide-antibody fusions was assessed using Homogenous Time Resolved Fluorescence (HTRF) epitope competition assays. In these assays a fluorescence resonance energy transfer (FRET) complex is formed between Streptavidin Cryptate, biotinylated-PCSK9 and a fluorescently (DyLight 650) labelled anti-PCSK9 antibody. Unlabelled peptide-antibody fusions that bind the same or overlapping epitopes of PCSK9 as that bound by the fluorescently labelled antibody will compete resulting in a reduction in FRET signal.

Labeling of the anti-PCSK9 antibodies was carried out using a DyLight-650 (Thermo Scientific 84536) according to the manufacturer instructions. For the assay all samples and reagents were prepared in assay buffer containing 1X Phosphate Buffered Saline, 0.1% BSA (Sigma A9576) and 0.4M Potassium Fluoride. Test samples of peptide-antibody fusions or control antibodies were prepared in 384 well polypropylene plates by 3-fold serial dilutions. Samples (5 μl ) or assay buffer (total binding control wells) were transferred to a 384-well assay plate (Costar 3676) and incubated for 4 hours at room temperature with 1 nM Streptavidin Cryptate (Cisbio 61SAXLB), 0.05-0.5 nM biotinylated-PCSK9 and 0.1-2 nM Dy650 labelled anti PCSK9 antibody in a 20 μl total assay volume. Non-specific binding (NSB) control wells were set up with the biotinylated PCSK9 omitted. Time resolved fluorescence emission at 665 nm and 620 nm was measured following excitation at 320 nm using the Perkin Elmer Envision. The ratio of the 665 nm counts/620 nm counts was calculated and multiplied by 10000 to give HTRF Counts. Delta F% was then calculated using the following equation.

${{Delta}\mspace{14mu} F\mspace{14mu} \%} = {\left( \frac{{{Sample}\mspace{14mu} H\; T\; R\; F\mspace{14mu} {Counts}} - {N\; S\; B\; H\; T\; R\; F\mspace{14mu} {Counts}}}{N\; S\; B\mspace{14mu} H\; T\; R\; F\mspace{14mu} {Counts}} \right) \times 100}$

The results were expressed as % specific binding according to the following equation:

${\% \mspace{14mu} {Specific}\mspace{14mu} {Binding}} = {\left( \frac{\left( {{{Sample}\mspace{14mu} {Delta}\mspace{14mu} F\mspace{14mu} \%} - {N\; S\; B\mspace{14mu} {Delta}\mspace{14mu} F\mspace{14mu} \%}} \right)}{\left( {{{Total}\mspace{14mu} {Binding}\mspace{14mu} {Delta}\mspace{14mu} F\mspace{14mu} \%} - {N\; S\; B\mspace{14mu} {Delta}\mspace{14mu} F\mspace{14mu} \%}} \right)} \right) \times 100}$

FIGS. 2A-G and FIGS. 3A-G showing the inhibition of human PCSK9 binding to anti-PCSK9 antibodies using a titration of unlabelled heavy chain or light chain peptide antibody fusions respectively. Isotype match irrelevant antibody NIP228 IgG1-TM alone (Control#1) or in fusion to the heavy (Control#2) or light chain (Control#3) with a GLP-1 analogue peptide of SEQ ID NO:28 using a linker of SEQ ID NO:4 were used as negative controls.

Potency of each peptide antibody fusion at human GLP-1 receptor was assessed using a cAMP production assay. Stable cell lines expressing human GLP-1 receptor was generated in CHO cells by standard methods. GLP-1 receptor activation by tested compounds will result in downstream production of cAMP second messenger that can be measured in a functional activity assay. Low protein binding 384-well plates (Greiner) were used to perform eleven 1 in 4 serial dilutions of test compound that were made in assay medium (0.1% bovine serum albumin in Hanks Balanced Salt Solution (GIBCO or Sigma), containing 0.5 mM IBMX (Sigma)). All sample dilutions were made in duplicate. A frozen cryo-vial of cells expressing human GLP-1 receptor was thawed rapidly in a water-bath, transferred to pre-warmed assay media and spun at 240 xg for 5 minutes. Cells were then re-suspended in assay buffer at the optimized concentration of 1×10⁵ cells/mL and dispensed at 5 uL per well to black shallow-well u-bottom 384-well plates (Corning). 5 μL of test compound was transferred from the dilution plate to the cell plate and incubated at room temperature for 30 minutes. cAMP levels were measured the cAMP dynamic 2 HTRF kit (Cisbio), following the two step protocol as per manufacturer's recommendations. Briefly, anti-cAMP cryptate (donor fluorophore) and cAMP-d2 (acceptor fluorophore) were made up separately by diluting each 1/20 in conjugate & lysis buffer provided in the kit. 5 uL of anti-cAMP cryptate was added to all wells of the assay plate and 5 uL of cAMP-d2 added to all wells except non-specific binding wells, to which only conjugate and lysis buffer was added. Plates were incubated at room temperature for one hour and then read on an Envision (Perkin Elmer) using excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. Data was transformed to % Delta F as described in manufacturer's guidelines and analyzed by unconstrained 4-parameter logistic fit of data, curve mid-point to determine EC₅₀.

Activation of the human GLP-1 receptor by heavy chain (A) or light chain (B) peptide antibody fusions are shown in FIGS. 4A-B, respectively. Free human GLP-1 peptide (Bachem) and irrelevant isotype match NIP228 human IgG1-TM without peptide were used as positive and negative controls respectively.

To conclude, all tested fusions were able to compete with anti-PCSK9 antibodies for binding to human PCSK9 as well as activating the human GLP-1 receptor. Those fusion molecules between a GLP-1 analogue peptide and different anti-PCSK9 antibodies display dual activity and can be used to provide combined pharmacology.

Example 4 In Vivo Stability of Existing GLP-1 Analogues in Antibody Fusion

Without target-mediated clearance, human IgGs have a long circulating half-life in man of around 21 days. This is notably due to the rescue of internalised antibodies by FcRn receptor. After non-specific uptake by cells, antibodies can bind to FcRn in the acidic environment of endosomes and be directed to the cell surface and back into circulation rather than to lysosomes for degradation.

In order to achieve maximum efficacy, GLP-1 analogue peptide in fusion with anti-PCSK9 antibody molecules should display adequate in vivo activity half-life for both activation of the GLP-1 receptor and PCSK9 suppression mediated by the peptide and the antibody moieties respectively. For instance if the peptide is quickly inactivated after injection, a majority of the product will only be functional for PCSK9 suppression and will not properly engage with the GLP-1 receptor over time to provide efficient glucose control through the dosing period.

In order to assess in vivo stability of existing GLP-1 analogue peptides when in antibody fusion, the following fusions were generated:

1: A GLP-1 analogue peptide of SEQ ID NO:28 was fused using a linker of SEQ ID NO:4 to the heavy chain of the irrelevant NIP228 human IgG1-TM antibody of SEQ ID NO: 414 and 415 (compound NIP228_GLP-1_VH).

2: Exendin-4 peptide, a GLP-1 analogue derived from Gila monster' saliva, of SEQ ID NO:12 was fused using a linker of SEQ ID NO:4 to the light chain of the anti-PCSK9 antibody PC9#2 of SEQ ID NO 9 (compound PC9#2_Exe4_VL).

3: A GLP-1 analogue peptide of SEQ ID NO:28 was fused using a linker of SEQ ID NO:4 to human IgG4 Fc fragment of SEQ ID NO: 416 (compound GLP-1-Fc)

4: A GLP-1 analogue peptide of SEQ ID NO:28 was fused using a linker of SEQ ID NO:4 to the light chain of the anti-PCSK9 antibody PC9#2 of SEQ ID NO 9 (compound PC9#2_GLP-1_VL).

All compounds are active at the human GLP-1 receptor in vitro and display EC₅₀ in the cAMP assay described in Example 3 of 2.08E-10 M,1.12E-10 M, 1.12E-10 M and 1.03E-10 M for NIP228_GLP-1_VH, PC9#2_Exe4_VL, GLP-1-Fc and PC9#2_GLP-1_VL respectively.

Compounds NIP228_GLP-1_VH and PC9#2_Exe4_VL were injected intravenously to rat and serum or plasma samples were collected at several timepoints post-injection. Compound concentration in serum or plasma (exposure) and concentration in active compound for GLP-1 activity were determined for each sample. Comparison between decline over time of total compound (exposure) and active compound at GLP-1 receptor provides an assessment of the in vivo stability for the GLP-1 analogue peptide in antibody fusion.

Levels of total human IgG1 antibody in rat plasma or serum were quantified by a generic sandwich enzyme-linked immunosorbent assay (ELISA) method using the Gyrolab platform (Gyros AB). Human IgG1 was captured by a biotinylated monoclonal anti-human IgG1 antibody (clone JDC-10, Southern Biotech, for plasma samples or in-house clone TM446 for serum samples) at 100 ug/mL and detected by an Alexa-labelled monoclonal anti-human IgG1 antibody at 25 nM (BD Pharmingen clone G18-145 for plasma samples) or 10 nM (the Binding Site AU003CUS01 for serum samples) on Gyrolab Bioaffy 200 CD. Standards, controls, plasma or serum samples, wash solution, capture and detection antibodies were added to a 0.2 mL 96-well PCR plate (Thermo Scientific) according to the Gyrolab method and loaded onto the machine with the CD200 plate. All samples were analyzed in duplicate. The mean response of each human IgG standard was plotted against concentration and the points were fit using a 5-parameter weighted logistic model using the Gyrolab Evaluator software.

The concentration of active peptide-antibody fusion at human GLP-1 receptor in rat samples was estimated using an ex-vivo cAMP cell based assay. Reference compounds were spiked into naïve rat serum or plasma at a known concentration to be used as a standard. All samples were serially diluted in assay medium and examined using the cAMP dynamic 2 HTRF kit (Cisbio) as described in Example 3 for serum samples or using the LANCE® Ultra cAMP Detection Kit (Perkin Elmer) for plasma samples.

Test samples were plotted using the same top concentration as the equivalent reference. The EC₅₀ values obtained could then be used to calculate the Sample Ratio (Sample EC₅₀/EC₅₀ reference compound) and then the estimated concentration in active GLP-1 compound (known top concentration of reference compound spiked into rat plasma or serum/Sample Ratio). Rat serum or plasma alone has a quenching effect on the cryptate donor signal and gives concentration-dependent activation of cAMP that can be diluted out. Any tested compound must therefore possess cAMP activity above that of the rat serum or plasma baseline (termed the limit of detection) in order to have an observable effect in the activity assay.

Compound NIP228_GLP-1_VH was injected at 2 mg/kg in three Wistar rats (Charles River) and blood samples for each animal were collected in EDTA tubes containing dPP4 inhibitor at 2 minutes, 1 h, 24 h, 48 h, 120 h, 168 h and 216 h post injection. Total compound and concentration in active GLP-1 compound in rat plasma over time are shown in FIGS. 5A and 9. Concentration of active compound in samples collected after 48 h cannot be determined as they were below the lower limit of quantification of the assay.

Compound PC9#2_Exe4_VL was injected at 1 mg/kg in three CD rats (Charles River) and blood samples were collected in plain tubes containing dPP4 inhibitor at 30 minutes, 6 h, 24 h, 48 h, 96 h, 240 h and 336 h post injection. Serum samples were prepared by leaving the tubes on the bench for 30 minutes followed by 2 minutes centrifugation at 13000 rpm. Exposure and concentration in active GLP-1 compound in rat serum over time are shown in FIG. 5B. Concentration of active compound in samples collected after 48 h cannot be accurately determined as they were below the lower limit of quantification of the assay.

In vivo half-life in rat of NIP228_GLP-1_VH and PC9#2_Exe4_VL for both exposure and active compound at GLP-1 receptor are presented in Table 9.

TABLE 9 Compound and active GLP-1 in vivo half-life in rat of existing GLP-1 analogues in antibody fusion Compound Active GLP-1 Experimental in vivo half in vivo half Compound design life (h) life (h) NIP228_GLP-1_VH 2 mg/kg IV 63 7.4 in Wistar rats PC9_2_Exe4_VL 1 mg/kg IV 88 5.7 in CD rats

For both NIP228_GLP-1_VH and PC9#2_Exe4_VL compounds, activity at GLP-1 receptor is loss much quicker than the compound itself demonstrating in vivo peptide instability.

Compounds GLP-1-Fc and PC9#2_GLP-1_VL were injected intravenously to healthy C57/B6 mice (7-8 weeks old, females, Charles River) and plasma samples were collected at several timepoints. Compound concentration in plasma (exposure) and concentration in active compound for GLP-1 activity were determined for each sample as described above.

GLP-1-Fc was injected at 1 mg/kg. Groups of three mice were sacrificed at each of the following time points: pre-injection, 2 minutes, 1 h, 6 h, 24 h, 48 h, 72 h and 96 h post injection. Blood for each animal were collected in EDTA tubes containing dPP4 inhibitor. Plasma samples were then centrifuged at 14000 rpm for 5 min at 4° C. and stored at −80° C. pending analysis.

PC9#2_GLP-1_VL was injected at 5 mg/kg and mice were sacrificed at each of the following time points: pre-injection, 5 minutes, 6.5 h, 24 h, 72 h and 168 h post injection. Samples were treated as described above.

Exposure and concentration in active GLP-1 compound in mouse plasma over time for GLP-1-Fc are shown in FIGS. 5C and 14A and PC9#2_GLP-1_VL are shown in FIGS. 5D and 10, respectively.

In vivo half-life in mice of GLP-1-Fc and PC9#2_GLP-1_VL for both exposure and active GLP-1 are presented in Table 10.

TABLE 10 Compound and active GLP-1 in vivo half-life in mice of existing GLP-1 analogues in antibody or Fc fusion Compound Active GLP-1 Experimental in vivo half in vivo half Compound design life (h) life (h) GLP-1-Fc 1 mg/kg in ~100 16 C57/B6 mice PC9#2_GLP-1_VL 5 mg/kg in ~100 18 C57/B6 mice

As observed for NIP228_GLP-1_VH and PC9#2_Exe4_VL in rat, activity at GLP-1 receptor for both GLP-1-Fc and PC9#2_GLP-1_VL following injection in mice is loss quicker than the compound itself, demonstrating in vivo peptide instability.

Quick inactivation for both GLP-1 analogues of SEQ ID NO:28 and SEQ ID NO:12 will impact efficient glucose control for PCSK9/GLP-1 fusion molecules and GLP-1 analogue peptides with better in vivo stability need to be engineered.

Example 5 Affinity for huPCSK9

The PCSK9 antibody was used as a benchmark control. Data are presented in Table 11 and a visual representation of the PC9#2_GLP1 molecule and the anti-PC9#2 antibody used as a benchmark control are shown in FIG. 8. A visualization of a potential target profile to guide PCSK9 affinity and GLP-1 potency is provided in FIG. 6.

Association (ka or kon), dissociation (kd or koff) and equilibrium dissociation constants (KD) for PCSK9 binding were determined at 25° C. by Surface Plasmon Resonance (SPR) using the Biacore 2000 biosensor (GE Healthcare).

A Protein G surface was first created on a CM5 sensor chip (GE Healthcare). Human antibodies were then captured on the chip surface before injecting different concentrations of human, cynomolgus or rat PCSK9. Global dissociation rates were first calculated followed by global on-rate calculations both using a 1:1 binding kinetics model.

TABLE 11 Affinity for huPCSK9 (Biacore) Test Compound Kd (nM) _(kon) (M⁻¹ · s⁻¹) k_(off) (s⁻¹) PC9#2 (SEQ ID 7 7.8E+04 5.5E−04 NO. 8 & 9) PC9#2_GLP1 (SEQ 19 2.7E+04 5.0E−04 ID NO: 8, 9, 4, & 28) (with the light chain fusion SEQ ID NO: 43) x change 2.7 2.9 0.9

This demonstrates that the fusion is only marginally impacting PCSK9 binding.

Additionally, the dual action fusion molecule HS9_DSB7 was tested in a Biacore assay as described in Example 17 to determine its affinity for human PCSK9. The PCSK9 antibody alone (PC9_2_FG_HS#9 of SEQ ID 1 and 2 for heavy and light chain respectively) was used as a benchmark control. Table 12 provides the data. This data demonstrates that the fusion is only marginally impacting PCSK9 binding across species (human (Hu), cynomolgus monkeys (Cy) and rat).

TABLE 12 Affinity for huPCSK9, CyPCSK9, and RatPCSK9 (Biacore) Hu Cy Rat ka kd KD ka kd KD ka kd KD Summary (M⁻¹ · s⁻¹) (s⁻¹) (M) (M⁻¹ · s⁻¹) (s⁻¹) (M) (M⁻¹ · s⁻¹) (s⁻¹) (M) PC9_2_FG_HS#9 3.47E+05 3.65E−04 1.05E−09 3.19E+05 3.74E−04 1.17E−09 9.69E+05 3.45E−04 3.56E−10 HS9_DSB7 1.46E+05 2.87E−04 1.97E−09 9.49E+04 2.84E−04 2.99E−09 5.07E+05 2.67E−04 5.27E−10

Example 6 GLP-1 Potency in cAMP Cell-Based Assay

PC9#2_GLP-1 was tested in a cAMP cell-based assay to determine its potency as described in Example 3. The GLPI peptide alone was used as a control and GLP-1Fc was used as a benchmark. Data are presented in Table 13 and a visual representation of the PC9#2_GLP1 molecule and the GLP-1 Fc used as a benchmark control are shown in FIG. 8.

TABLE 13 GLP-1 Potency in cAMP Cell-Based Assay Test Compound EC50 (pm) x change PC9#2_GLP1 (SEQ 290 2.4 ID NO: 8, 9, 4, & 28) GLP1-Fc ( 120 1 GLP1 peptide (SEQ 15 0.1 ID NO: 29)

This data demonstrates that there was no significant loss of GLP-1 potency compared to the benchmark molecule GLP-1-Fc following the fusion of the GLP-1 analogue peptide to the light chain of the anti-PCSK9 antibody PC9#2.

Example 7 Dulaglutide: A Benchmark Molecule

FIG. 14A provides stability in rat of GLP-1-Fc benchmark. Benchmark molecule is a GLP-1 moiety fusion to an Fc portion of an antibody, as shown in FIG. 14A.

Compounds GLP-1-Fc was injected intravenously to healthy C57/B6 mice (7-8 weeks old, females, Charles River) and plasma samples were collected at several timepoints. Compound concentration in plasma (exposure) and concentration in active compound for GLP-1 activity were determined for each sample as described in Example 4.

GLP-1-Fc was injected at 1 mg/kg. Groups of three mice were sacrificed at each of the following time points: pre-injection, 2 minutes, 1 h, 6 h, 24 h, 48 h, 72 h and 96 h post injection. Blood for each animal were collected in EDTA tubes containing dPP4 inhibitor. Plasma samples were then centrifuged at 14000 rpm for 5 min at 4° C. and stored at −80° C. pending analysis.

GLP-1 activity is lost at a quicker rate than the compound, demonstrating peptide instability. The line with the squares corresponds to the serum concentration of GLP-1-Fc and the line with the circles corresponds to the activity of GLP-1-Fc for the same samples. See FIG. 14A.

Example 8 Fusion Molecules with Enhanced In Vivo Stability Profiles

A) Evaluating GLP-1 Analogue Peptides in Antibody Fusion with Enhanced In Vivo Stability Profiles

To improve in vivo peptide stability of antibody fusion molecules, steric hindrance was engineered around the peptide to protect it from degradation. This was done either by introducing a bulky sugar motif or by engineering an inter molecular disulphide bridge.

It has been demonstrated that addition of N-glycosylation consensus motifs can increase in vivo stability and duration of action of proteins (Elliott S. et al., Nat. Biotech., 2003, 21, 414-421). GLP-1 analogue peptides incorporating an extra N-glycosylation motif, NxS or NxT where x can be any amino acid except proline, at the C-terminus of the peptide or in the linker between the peptide and the antibody have been engineered in fusion with the light chain of anti-PCSK9 antibody PC9#2 (antibody light chain, SEQ ID NO: 9). Peptide and linker amino acid sequence for eight of those compounds (named NGS for N-Glysosylation Site) are shown in FIG. 7A. Amino acid changes to generate the glycosylation motif are shown in bold underlined.

Among those eight compounds, only PC9_2_GLP-1_NGS#7 shows a high glycosylation yield by SDS-PAGE. This was detected by an increase in the molecular weight of the light chain compared to the control compound without the glycosylation consensus sequence, and with no visible lower molecular band corresponding to the non-glycosylated light chain product. Glycosylation for PC9_2_GLP-1_NGS#7 was further confirmed by ESI mass spectrometry.

It has also been shown that introducing an inter-disulphide bond could be a successful approach to improve in vivo stability of GLP-1 analogues as free peptide (Li Y. et al., Peptides, 2011, 21, 1303-1312).

Exendin-4 peptide variants incorporating two cysteine residues to form the disulphide bridge as well as, if appropriate, a glycine C-terminus cap in order to facilitate the bonding were fused to the light chain of anti-PCSK9 antibody PC9#2 (SEQ ID NO: 9). Peptide amino acid sequence for the three compounds initially generated (named DSB for DiSulphide Bridge) are shown in FIG. 7B (as SEQ ID NOs: 30-32). Cysteine residues are shown in black, other mutated residues are shown as underline and additional glycine residues at the C-terminus cap are shown in grey.

For DSB#1 variant, the first cysteine was engineered in position 9 instead of an aspartic acid and using a C-terminus cap, incorporating the second cysteine, of sequence:

(SEQ ID NO: 401) GGGGGGGGGGGCGG.

For DSB#2 variant, the first cysteine was engineered in position 4 instead of a glycine and using a C-terminus cap, incorporating the second cysteine, of sequence:

(SEQ ID NO: 402) GGGGGGGGGGGGCG.

For DSB#3 variant, the first cysteine was engineered in position 18 instead of an alanine but no C-terminus cap was used. The second cysteine was introduced at position 39 of the Exendin-4 sequence instead of a serine. Proline 38 was also changed into a glycine to generate more flexibility in the tryptophan cage of Exendin-4 in order to facilitate the formation of the disulphide bridge.

PC9_2_Exe4_DSB#2 in light chain fusion does not express significantly in mammalian cells and was not further characterised but sufficient amount of PC9 2 Exe4 DSB#1 and PC9_2_Exe4_DSB#3 were obtained. Integrity and identity of the fusions were confirmed by ESI mass spectrometry before in vivo experiments.

In vivo stability of PC9_2_GLP-1_NGS#7, PC9_2_Exe4_DSB#1 and PC9_2_Exe4_DSB#3 was assessed in mouse by following both compound exposure and concentration in active GLP-1 over time as described in Example 4.

Healthy C57/B6 mice (7-8 weeks old, females, Charles River) received one single intravenous (IV) dose of PC9_2_GLP-1_NGS#7 at 40 mg/kg, PC9_2_Exe4_DSB#1 at 10.8 mg/kg or PC9_2_Exe4_DSB#3 at 5 mg/kg. Groups of three mice were sacrificed at each of the following time points: pre-injection, 5 minutes, 6 h, 24 h, 72 h and 168 h post injection and blood for each animal were collected in EDTA tubes containing dPP4 inhibitor. Plasma samples were then centrifuged at 14000 rpm for 5 min at 4° C. and stored at −80° C. pending analysis.

All three compounds are active at the human GLP-1 receptor in vitro but display different EC₅₀ in the cAMP assay: 1.94⁻⁸M, 5.25 ⁻⁹ M and 3.25 ⁻¹⁰M for PC9_2_GLP-1_NGS#7, PC9_2_Exe4_DSB#1 and PC9_2_Exe4_DSB#3 respectively. Doses were adjusted as much as possible based on potency to generate a signal above the lower limit of quantification in the cAMP ex-vivo assay in order to calculate concentration in active GLP-1 compound over a significant period of time.

Exposure and concentration in active GLP-1 compound in mouse plasma over time for PC9_2_GLP-1_NGS#7 are shown in FIGS. 12 and 13A, PC9_2_Exe4_DSB#1 are shown in FIGS. 13B and 14B, and PC9_2_Exe4_DSB#3 fusion molecules are shown in FIGS. 11 and 13C, respectively.

In vivo half-life of PC9_2_GLP-1_NGS#7, PC9_2_Exe4_DSB#1 and PC9_2_Exe4_DSB#3 for both exposure and active GLP-1 are presented in Table 14.

TABLE 14 Compound and active GLP-1 in vivo half-life of engineered GLP-1 analogues in antibody fusion Compound Active GLP-1 Experimental in vivo in vivo Compound design half life (h) half life (h) PC9_2_GLP- 40 mg/kg IV in ~100 76 1_NGS#7 C57/B6 mice PC9_2_Exe4_DSB#1 10.8 mg/kg IV ~100 ~100 in C57/B6 mice PC9_2_Exe4_DSB#3 5 mg/kg IV in ~100 36 C57/B6 mice

Compared to parent molecules NIP228_GLP-1_VH and PC9#2_Exe4_VL (Table 9), all three compounds have improved in vivo stability for GLP-1 activity with half-life of 76 h, around 100 h and 36 h for PC9_2_GLP-1_NGS#7, PC9_2_Exe4_DSB#1 and PC9_2_Exe4_DSB#3 respectively.

Quite interestingly, PC9_2_Exe4_DSB#1 appears fully stable in mice for up to 7 days with no observed loss of GLP-1 activity when compared to compound exposure (FIG. 13B).

Such data are suggesting that generating steric hindrance around GLP-1 analogues can increase in vivo activity half-life of the peptide in antibody fusion.

B) Evaluating Fusion Molecules with Enhanced In Vivo Stability Profiles

The protocol discussed in Example 4 and 8A was followed to administer various compounds to mice and to plot the concentration of the compounds in the plasma over time. Compound potency (EC50) at human GLP-1 receptor was determined using the cAMP assay as described in Example 3.

Fusion molecule optimization was guided through an in vivo PK/stability assessment. As shown in FIGS. 14A, 11 and 12, peptide engineering produced fusion molecules with enhanced in vivo stability profiles. Benefits were seen with disulfide bridge stabilization for which there was longer retention of activity with minimum impact on potency at GLP-1 receptor. NGS#7 also has an improved stability profile but a low potency (19 nM) compared to DSB#3 (340 pM).

FIG. 14A shows a GLP-1-Fc benchmark molecule with an EC₅₀ of 100 pm.

FIG. 11 shows a disulfide bridged variant (PC9_2_DSB#3) (SEQ ID NO: 49 and SEQ ID NO: 8) with an EC₅₀ of 340 pM.

FIG. 12 shows an n-glycosylation variant (PC9_2_NGS#7) (SEQ ID NO: 50 and SEQ ID NO: 8) with an EC₅₀ of 19 nM.

In vivo half-life for those compounds are presented in Table 15.

TABLE 15 Compound and active GLP-1 in vivo half-life of engineered GLP-1 analogues in antibody fusion Compound Active GLP-1 Experimental in vivo in vivo Compound design half life (h) half life (h) GLP-1-Fc 1 mg/kg in ~100 16 C57/B6 mice PC9_2_Exe4_DSB#3 5 mg/kg IV in ~100 36 C57/B6 mice PC9_2_GLP- 40 mg/kg IV in ~100 76 1_NGS#7 C57/B6 mice

Example 9 A PCSK9/GLP-1 Fusion Demonstrates an Ideal Stability/Activity Profile

The dual action fusion molecule was evaluated for Fc-exposure and GLP-1 activity in a mouse model. Dosing was 10.8 mg/kg intravenously and the in vitro potency was 5200 pM. Protocol was as described in Example 8. The data were compared to the GLP-1 Fc benchmark molecule, with dosing of 1 mg/kg intravenously and an in vitro potency of 100 pM. Similar protocol was here used as described in Example 4. Results are shown in FIGS. 14A (dulaglutide) and 14B (PCSK9/GLP-1 fusion PC9_2_DSB#1) (SEQ ID NO: 48 and SEQ ID NO: 8). There was no loss of GLP-1 activity in the mouse for up to 7 days.

Example 10 Production and Purification of Early GLP-1 Analogue Peptide Antibody Fusions with an Intramolecular Disulphide Bridge

Quality of the material post-protein A purification from medium scale batches was poor for PC9_2_Exe4_DSB#1 and PC9_2_Exe4_DSB#3 as lot of aggregates were detected by SEC-HPLC. Preparative Size Exclusion Chromatography using Superdex 200 prep grade columns was then used to further purify the compounds as described in Example 2.

Preparative SEC chromatograms for PC9_2_Exe4_DSB#1 and PC9_2_Exe4_DSB#3 are shown in FIGS. 15A and 15B, respectively. A significant proportion (25-40%) of the material is aggregated as shown by additional peaks at early retention time. Fractions containing the monomeric compound were collected to obtain the material used for in vivo testing. See Example 8 and 9.

Scalability of PC9_2_Exe4_DSB#1 production was further assessed by transiently transfected 48.2 L of CHO mammalian cells in wavebags (GE Healthcare). Compound was purified by using a Protein A column followed by two additional purification steps using a mixed-mode resin.

A high level of aggregation (>25%) was detected in the harvest and efficient purification of the monomer product was particularly challenging. Three chromatography steps were required to generate a product at 95.9% purity by SEC-HPLC. This was very detrimental to the purification yield with an overall recovery of around 3.4%. In addition, the titre in harvest was at 104 mg/L which is low compared to monoclonal antibodies using a similar expression system. Production of DSB#1 disulphide bridge GLP-1 analogue in fusion with the light chain of anti-PCSK9 antibody HS9 (SEQ ID NO: 2) gave very similar results.

Example 11 Aggregation

The dual action fusion molecule (PCSK9/GLP-1 fusion PC9_2_DSB#1) (SEQ ID NO: 48) has the potential for aggregation and in some embodiments monomer is desired to be selected. Aggregates, detected during an analytical SEC-HPLC following protein A purification as described in Example 2, are shown in FIG. 16. Thus, in some embodiments additional engineering may be desired to improve the monomeric profile.

Example 12 GLP-1 Analogue Peptides in Antibody Fusion with Enhanced Monomeric Profiles

Additional Exendin-4 disulphide bridge peptides (DSB) in fusion with the light chain of PC9#2 (SEQ ID NO: 9) were generated in order to improve the monomeric profile during production of the peptide antibody molecule. Different positions of the cysteine bridge as well as length of the glycine rich C-terminus cap and various glycine point mutations in the C-terminus of the Exendin-4 peptide were engineered in order to facilitate the formation of the disulphide bond and ultimately reduce aggregation during production probably due to disulphide scrambling. Peptide engineering work was guided using the 3-D NMR structure of Exendin-4 (Neidigh, J W et al., Biochemistry, 2001, 40, 13188-200.).

A total of ten DSB peptide anti-PCSK9 fusions were produced at small scale and screened for an improved monomeric profile by SEC-HPLC. Peptide sequences are described in FIG. 17. PC9_2_Exe4_DSB#4 did not significantly express and was not further characterised.

Percentages of aggregate for the nine fusions compared to PC9_2_Exe4_VL and PC9_2_Exe4_DSB#1 determined by analytical SEC-HPLC after protein A purification are described in Table 16.

TABLE 16 Percentage of aggregate post protein A purification for disulphide bridge exendin-4 variants in fusion with the light chain of PCSK9 antibody PC9_2 % aggregate by # Compound analytical SEC 1 PC9_2_Exe4_VL 2.9 2 PC9_2_Exe4_DSB#1 26.5 3 PC9_2_Exe4_DSB#5 20.5 4 PC9_2_Exe4_DSB#6 23.3 5 PC9_2_Exe4_DSB#7 5.1 6 PC9_2_Exe4_DSB#8 51.1 7 PC9_2_Exe4_DSB#9 7.8 8 PC9_2_Exe4_DSB#10 7.3 9 PC9_2_Exe4_DSB#11 7.6 10 PC9_2_Exe4_DSB#12 20.1 11 PC9_2_Exe4_DSB#13 17.1

Four compounds among the nine tested achieve a percentage aggregate below 10%: PC9_2_Exe4_DSB#7, PC9_2_Exe4_DSB#9, PC9_2_Exe4_DSB#10 and PC9_2_Exe4_DSB#11. PC9_2_Exe4_DSB#7 has the lowest percentage aggregate at 5.1% compared to 26.5% for the early fusion PC9_2_Exe4_DSB#1. Exendin-4 antibody fusion without a disulphide bridge, PC9_2_Exe4_VL, shows 2.9% aggregate.

Productions of PC9_2_Exe4_DSB#7 and PC9_2_Exe4_DSB#9 were scaled up to supply material in sufficient quantity to perform in vivo stability experiments. FIGS. 18 and 19 show showing preparative SEC chromatograms of PC9_2_Exe4_DSB#7 and PC9_2_Exe4_DSB#9 respectively following an initial protein A purification as described in Example 2. Unlike PC9_2_Exe4_DSB#1 and PC9_2_Exe4_DSB#3 (FIGS. 15A and B), no significant proportion of aggregates was detected during that purification step.

In order to check that the optimised DSB peptide antibody fusions with an improved monomeric profile do exhibit a superior in vivo stability compared to Exendin-4 antibody fusion, PC9_2_Exe4_DSB#7 and PC9_2_Exe4_DSB#9 were injected intravenously in three CD rats for each compound at 10 mg/kg and 1 mg/kg respectively. Serum samples for each animal were collected at 30 minutes, 6 h, 24 h, 48 h, 96 h, 240 h and 336 h post injection. Compound exposure and concentration in active GLP-1 were measured over time as described in Example 4.

Both compounds are active at the human GLP-1 receptor in the cAMP assay with EC₅₀ of 9.45E-10 M for PC9_2_Exe4_DSB#7 and 1.24E-10 M for PC9_2_Exe4_DSB#9. PC9_2_Exe4_DSB#7 is around eight time less potent than PC9_2_Exe4_DSB#9 and as thus been injected at ten time higher a dose to generate a signal above the lower limit of quantification in the cAMP ex-vivo assay over a significant period of time.

Exposure and concentration in active GLP-1 compound in rat serum over time for PC9_2_Exe4_DSB#7 and PC9_2_Exe4_DSB#9 fusion molecules are shown in FIGS. 20 and 21, respectively. Concentrations in active GLP-1 were normalised to the exposure at the first time point (30 min) to simplify the analysis.

PC9_2_Exe4_DSB#7 and PC9_2_Exe4_DSB#9 have a half-life for GLP-1 activity of 44.2 h and 12.5 h respectively compared to 5.7 h for the parent Exendin-4 fusion molecule PC9_2_Exe4_VL (see Table 9)). Concentrations of active compound for PC9_2_Exe4_DSB#9 samples collected after 96 h cannot be determined as they were below the lower limit of quantification of the assay.

Those data are demonstrating that both PC9_2_Exe4_DSB#7 and PC9_2_Exe4_DSB#9 have an improved in vivo activity half-life of the GLP-1 analogue peptide when in antibody fusion compared to the parent fusion molecule.

As described above peptide engineering can manage the aggregation profile. Mutations were made in the position of the cysteine bridge in the peptide and the composition and length of the peptide/C terminus of the GLP-1 moiety.

The protocol was as described above.

PCSK9/GLP-1 fusion (PC9_2_DSB#7) (SEQ ID NO: 51 and SEQ ID NO: 8) achieved >90% monomer by SEC-HLPC in small-scale batch. Aggregation was still detected in medium scale batch but to a much lower extended than PCSK9/GLP-1 fusion PC9_2_DSB#1 (SEQ ID NO: 48 and SEQ ID NO: 8).

Results are presented in FIGS. 15A, 18, 23 and Table 16.

Example 13 Pharmacokinetics and Pharmacodynamics Modeling of GLP-1 Analogue Peptides in Fusion with Anti-PCSK9 Antibodies

To guide the design of PCSK9/GLP-1 fusion molecules, a pharmacokinetic (PK) -pharmacodynamic (PD) model has been developed using prior data on the relationship between PCSK9 suppression and affinity of anti-PCSK9 antibodies tested in the clinic as well as data on the approved GLP-1 receptor agonist molecules Liraglutide and Dulaglutide.

The potency of GLP-1 analogue peptide in fusion with anti-PCSK9 antibody was scanned to identify the optimum range that would result in comparable GLP-1 activity to marketed drugs using a dose able to generate sufficient PCSK9 suppression. Simulations were performed using the pharmacokinetics, plasma protein binding, and receptor affinity properties of Dulaglutide to obtain the potency-normalised GLP-1 activity over time of that compound. For PCSK9/GLP-1 fusion molecule, the PK properties are assumed to be those of a typical human antibody directed towards PCSK9 and the information was derived from compounds in the clinic. These simulations indicate that a 60 mg subcutaneous weekly dose of PCSK9/GLP-1 fusions with an affinity of 3.9 nM for human PCSK9 should result in greater than 90% PCSK9 suppression over the dosing period at steady-state (FIG. 22A).

Using that dosing regimen, simulations indicate that potency of PCSK9/GLP-1 fusion molecules at human GLP-1 receptor should be within 3-5 nM in order to achieve similar GLP-1 activity compared to existing molecules (FIG. 22B). Potency of Dulaglutide in those simulations was set-up at 80 pM suggesting that potency of PCSK9/GLP1 fusion molecules need to be around 30 to 60 fold lower than Dulaglutide in order to manage nausea side effect associated with GLP-1 receptor agonist molecules at the dose required to efficiently suppress PCSK9.

Example 14 GLP-1 Analogue Peptides with Reduced Potency at the Human GLP-1 Receptor

Methods for reducing potency of the GLP-1 peptide or GLP-1 analogues are well known in the art as for instance mutating or introducing non-natural amino acids at key residues in the peptide. Critical residues for binding of GLP-1 peptide to the receptor and activity were notably identified by alanine scanning (Adelhorst, K. et al., J. Bio. Chem., 1994, 269, 6276-6278).

To further demonstrate the feasibility to reduce potency at GLP-1 receptor, a panel of GLP-1 analogues with a mutation or non-natural amino acids in position 2 or 3, using the 7-36 GLP-1 sequence, were synthesized as free peptide and tested for activity at the human GLP-1 receptor in the cAMP in vitro assay as described in Example 3. Data are summarised in Table 17 below. Aib is for 2-aminoisobutyric acid and Orn for ornithine.

TABLE 17 Potency at human GLP-1R in the cAMP assay of GLP-1 point mutant peptides Potency at huGLP- Fold difference 1R in cAMP assay in EC50 vs # Peptide Modification (nM) GLP-1 1 GLP-1 / 0.10 / 2 g769 E3D 0.10 1 3 g766 A2Aib 0.11 1.1 4 g770 A2P 0.15 1.5 5 g767 E3Q 0.28 2.8 6 g762 A2S 0.29 2.9 7 g755 A2H 0.32 3.2 8 g752 A2V 0.56 5.6 9 g749 A2G 0.63 6.3 10 g768 E3N 0.68 6.8 11 g751 A2I 1.78 17.8 12 g765 A2Q 2.98 29.8 13 g763 A2T 4.30 43 14 g753 A2F 4.30 43 15 g756 A2W 5.46 54.6 16 g754 A2Y 14.4 144 17 g764 A2N 21.0 210 18 g750 A2L 80.0 800 19 g761 A2Orn 86.4 864 20 g757 A2E 103.3 1033 21 g758 A2D >100 >1000 22 g760 A2R >80 >800 23 g759 A2K >100 >1000

Those data demonstrate that EC₅₀ of GLP-1 analogue peptides can be modulated to achieve a defined potency reduction.

Example 15 GLP-1 Analogue Peptides in Antibody Fusion with Reduced Potency at the Human GLP-1 Receptor

-   -   To reduce the potency of DSB#7 GLP-1 analogue peptide (SEQ ID         NO: 13) in fusion with the light chain of the anti-PCSK9         antibody HS9 (SEQ ID NO: 2), point mutations were introduced at         position G2, E15, V19, 123 or L26 in the peptide using standard         molecular biology techniques. A total of 28 mutants of the         peptide antibody fusion molecule were produced and tested for         activity at the human GLP-1 receptor using the cAMP assay as         described in Example 3. List of the compounds and activity data         are presented in Table 18.

TABLE 18 Potency at human GLP-1R and percentage maximum activation compared to GLP-1 of DSB#7 point mutants in light chain fusion of PCSK9 antibody HS9 Potency at Fold huGLP-1R difference SEQ in cAMP % Max in EC50 vs ID # Compound assay (M) Effect GLP1-Fc NO: 1 HS9_DSB7_G2Y 1.7E−08 10 603 444 2 HS9_DSB7_G2V 1.2E−09 93 43 445 3 HS9_DSB7_G2T 6.3E−09 73 227 446 4 HS9_DSB7_G2Q 1.2E−08 86 436 447 5 HS9_DSB7_G2N 1.9E−08 31 685 448 6 HS9_DSB7_G2I 2.3E−09 87 81 449 7 HS9_DSB7_G2F 5.8E−09 69 209 450 8 HS9_DSB7_E15G 6.8E−09 91 243 451 9 HS9_DSB7_E15A 1.2E−09 92 42 452 10 HS9_DSB7_V19T 4.0E−10 95 14 453 11 HS9_DSB7_V19S 2.7E−09 94 95 454 12 HS9_DSB7_V19G 1.3E−08 93 479 455 13 HS9_DSB7_V19A 1.3E−09 96 46 456 14 HS9_DSB7_I23T Inactive Inactive / 457 15 HS9_DSB7_I23S Inactive Inactive / 458 16 HS9_DSB7_I23G 5.6E−07 82 2.0E+04 459 17 HS9_DSB7_I23A 6.1E−07 67 2.2E+04 460 18 HS9_DSB7_L26T 1.3E−07 87 4.7E+03 461 19 HS9_DSB7_L26S Inactive Inactive / 462 20 HS9_DSB7_L26P 1.4E−06 60 5.1E+04 463 21 HS9_DSB7_L26N 4.3E−06 44 1.5E+05 464 22 HS9_DSB7_L26Q 5.9E−06 34 2.1E+05 465 23 HS9_DSB7_L26M 5.3E−08 90 1.9E+03 466 24 HS9_DSB7_L26I 7.4E−10 96 27 467 25 HS9_DSB7_L26H 8.0E−08 87 2.9E+03 468 26 HS9_DSB7_L26G 2.8E−06 81 1.0E+05 469 27 HS9_DSB7_L26E Inactive Inactive / 470 28 HS9_DSB7_L26D Inactive Inactive / 471 29 HS9_DSB7 2.1E−10 96 8 472 30 GLP1 1.9E−11 100  0.7 31 GLP1-Fc 2.8E−11 99 1

Five out of the twenty-eight constructs are inactive. The others compounds display very diverse potency at human GLP1 receptor ranging from 400 pM for HS9_DSB7_V19T to almost 6uM for HS9_DSB7_L26Q. In addition some compounds, as HS9_DSB7_G2Y or HS9_DSB7_L26Q, are partial agonists: they do not provide the same level of activation at saturating dose compared to GLP-1 peptide.

Based on PKPD modeling in Example 13 the dual activity anti-PCSK9 antibody GLP-1 receptor agonist molecules need to have a potency reduction at human GLP-1 receptor compared to the GLP-1-Fc benchmark of around 30 to 60 fold in order to manage nausea at the dose required to efficiently suppress PCSK9 antigen.

Four peptide antibody fusions (HS9_DSB7_G2V, HS9_DSB7_E15A, HS9_DSB7_V19A and HS9_DSB7_L26I) display a potency between 700 pM and 1.4 nM corresponding to a 25 to 50 fold loss compared to the benchmark GLP1-Fc. All those four compounds are full agonists at human GLP-1 receptor with a percentage of maximum activation greater than 90% compared to the GLP-1 peptide.

Example 16 Characterisation of GLP-1 Analogue Peptides in Fusion with Anti-PCSK9 Antibody

A) Developability Assessment of GLP-1 Analogue Peptides in Fusion with Anti-PCSK9 Antibody

The four selected peptide antibody fusions at the desired human GLP-1R potency (HS9_DSB7_G2V, HS9_DSB7_EISA, HS9_DSB7_V19A and HS9_DSB7_L26I) were produced in large scale to support further characterisation. To assess propensity of the compounds to aggregate during production, post protein A purification samples were tested by analytical SEC-HPLC. Data are summarised in Table 19.

TABLE 19 SEC-HPLC analysis of peptide/antibody lead molecules following Protein A purification # Compound % Aggregate % Monomer % Truncate 1 HS9_DSB7_G2V 25.08 74.64 0.28 2 HS9_DSB7_E15A 21.04 78.6 0.36 3 HS9_DSB7_V19A 4.33 95.43 0.24 4 HS9_DSB7_L26I 5.96 93.81 0.23

Only two out of the four fusions (HS9_DSB7_V19A and HS9_DSB7_L26I) achieve a percentage monomer greater than 90% post protein A purification. HS9_DSB7_V19A presents the best profile with more than 95% monomer. HS9_DSB7_G2V and HS9_DSB7_E15A are significantly prone to aggregation during production with percentage aggregate greater than 20% compared to less than 5% for HS9_DSB7_V19A.

Purified compounds were concentrated using centrifugal spin concentrators with a molecular weight cut-off of 30 kDa to achieve a target concentration of 50 mg/mL in default formulation buffer. Concentration of HS9_DSB7_G2V and HS9_DSB7_E15A was stopped at 38.7 and 33.7 mg/mL respectively as it was noticed that further volume reduction leads to a drop in protein concentration. No such issue was observed during the concentration step of HS9_DSB7_V19A and HS9_DSB7_L26I.

Samples were then incubated at 5° C. or 40° C. for 4 weeks followed by analytical SEC-HPLC in order to assess storage stability. Results are summarized in Table 20.

TABLE 20 Purity and aggregation parameters for purified peptide/antibody lead molecules after 4 weeks incubation at 5° C. or 40° C. Purity after Purity after Aggregation Aggregation [C] 4 weeks at 4 weeks at rate per month rate per month # Compound mg/mL 5° C. (%) 40° C. (%) at 5° C. (%) at 40° C. (%) 1 HS9_DSB7_G2V 38.7 * 88.6 77.9 1.4 9.2 2 HS9_DSB7_E15A 33.7 * 93.9 80.8 0.81 13.2 3 HS9_DSB7_V19A 52 98.7 90.1 0.3 6.3 4 HS9_DSB7_L26I 47.2 97 87.6 0.32 7.1

Both HS9_DSB7_V19A and HS9_DSB7_L26I display better stability parameters than HS9_DSB7_G2V and HS9_DSB7_E15A. For instance, the first two have an aggregation rate per month at 5° C. of around 0.3% compared to 0.8 and 1.4% for HS9_DSB7_E15A and HS9_DSB7_G2V respectively.

B) Single Intravenous Dose Pharmacokinetics in Rat of GLP-1 Analogue Peptides in Fusion with Anti-PCSK9 Antibody

Pharmacokinetic profile of the four selected peptide antibody fusions at the desired human GLP-1R potency was assessed as described in Example 4 following a single intravenous bolus in three CD rats at 60, 53, 58.5 and 60 mg/kg for HS9_DSB7_G2V, HS9_DSB7_E15A, HS9_DSB7_V19A and HS9_DSB7_L26I respectively. A high dose of the compound (above 50 mg/kg) was used to saturate the PCSK9 sink for a significant period of time to determine PK parameters during the linear phase, without any target mediated drug disposition component.

Blood samples were collected at 30 minutes, 6 h, 24 h, 48 h, 96 h, 168 h, 240 h and 336 h post injection. Concentrations for the four compounds over time are shown in FIG. 24 and half-life data are summarized in Table 21.

TABLE 21 In vivo half-life of peptide/antibody lead molecules in rats after a single i.v. injection. Exposure half-life # Compound Design (h) 1 HS9_DSB7_G2V 60 mg/kg iv in CD rats 48 2 HS9_DSB7_E15A 53 mg/kg iv in CD rats 41 3 HS9_DSB7_V19A 58.5 mg/kg iv in CD 128 rats 4 HS9_DSB7_L26I 60 mg/kg iv in CD rats 39

The four fusions molecules have significant different profiles despite being very close in sequence and sharing the same antibody backbone. HS9_DSB7_V19A has the longest in vivo half-life of the four fusions, 128 h compared to for instance only 39 h for HS9_DSB7_L26I.

Example 17 Affinity and Kinetic Parameters Determination for PCSK9 Across Species of GLP-1 Analogue Peptide in Fusion with Anti-PCSK9 Antibody

Association (ka), dissociation (kd) and equilibrium dissociation constants (KD) for human, cynomolgus and rat PCSK9 binding to the GLP-1 analogue anti-PCSK9 fusion HS9_DSB7_V19A human IgG1-TM were determined at 25° C. by Surface Plasmon Resonance (SPR) using the Biacore 2000 biosensor (GE Healthcare), essentially as described by Karlsson et al. (J. Immunol. Methods (1991), vol. 145, p. Dear229-40). Anti-PCSK9 antibody PC9#3 human IgG1-TM was used as benchmark (Variable heavy chain of SEQ ID NO: 404 and variable light chain of SEQ ID NO: 405.

A mouse anti-human IgG monoclonal antibody surface was first created using a Human Antibody Capture Kit and CM5 sensor chip (GE Healthcare). Human antibody compounds were captured at a flow rate of 10 μL/minute for 3 minutes. Recombinant human Avi_PCSK9_Flag_His (in-house), cynomolgus Avi_PCSK9_Flag_His (in-house) and His-tagged rat PCSK9 (SinoBiological) were diluted to concentrations ranging from 1 nM to 200 nM in running buffer (10 mM sodium phosphate pH 7.4, 150 mM sodium chloride, 1 mg/mL BSA, 0.05% Tween20) and injected over the chip surface for 10 minutes, followed by running buffer only for a 10 minutes dissociation phase. The surface of the chip was regenerated using 3 M magnesium chloride between each antibody application. Global dissociation rates were first calculated followed by global on-rate calculations both using a 1:1 binding kinetics model.

Results are shown in Table 22.

TABLE 22 Kinetic parameters determined by Biacore of peptide/antibody lead molecule HS9_DSB7_V19A for human, cynomolgus and rat PCSK9 compared to anti-PCSK9 antibody PC9#3 kinetic parameters ka (1/Ms) kd (1/s) KD (M) Human PCSK9 PC9#3 6.4E+05 4.4E−04 7.0E−10 HS9_DSB7_V19A 1.9E+05 1.1E−04 6.0E−10 Cynomolgus PCSK9 PC9#3 4.2E+05 4.2E−04 1.0E−09 HS9_DSB7_V19A 1.1E+05 1.9E−04 1.7E−09 Rat PCSK9 PC9#3 9.0E+05 4.7E−03 5.2E−09 HS9_DSB7_V19A 3.7E+05 2.1E−04 5.7E−10

Fusion molecule HS9_DSB7_V19A has an affinity at pH7.4 for human PCSK9 of 600 pM very similar to the benchmark anti-PCSK9 antibody PC9#3. Interestingly HS9_DSB7_V19A, with a four time lower dissociation constant, is less prone to dissociate from human PCSK9 compared to PC9#3.

In addition, HS9_DSB7_V19A can strongly bind to cynomolgus and rat PCSK9 at physiological pH with equilibrium dissociation constants close to the human PCSK9 value (1.7 nM and 570pM respectively).

Example 18 Specificity for PCSK9 Compared to Closely Related Human Proteins of GLP-1 Analogue Peptide in Fusion with Anti-PCSK9 Antibody

Specificity for PCSK9 compared to related human proteins of the GLP-1 analogue anti-PCSK9 fusion HS9_DSB7_V19A human IgG1-TM was determined by Dissociation-Enhanced Lanthanide Fluorescent Immunoassay Time Resolved Fluorescence (DELFIA TRF Assay, PerkinElmer). Recombinant human Avi_PCSK9_Flag_His (in-house), GST tagged human PCSK7 (Abnova), GST tagged human MBTPSI (Abnova) and Flag/His tagged human CD86 (in-house) were coated at 10 μg/mL in PBS into 96-well immunoassay plate. After washing, HS9_DSB7_V19A was added to antigen-coated wells at a concentration of 25 μg/mL. Plates were incubated at room temperature for 2 hours before extensive washing. Bound HS9_DSB7_V19A human IgG1-TM was detected using secondary Europium-labelled anti-human IgG antibody (PerkinElmer). Antigen coating to the plates was assessed by using a mouse anti-GST IgG (Abcam) as primary and Europium-labelled anti-mouse IgG (PerkinElmer) as detection for human PCSK7 and human MBTPSI. Human PCSK9 and human CD86 coating was directly assessed using a Europium-labelled anti-His IgG (PerkinElmer).

Results showed that HS9_DSB7_V19A human IgG1-TM binds strongly to human PCSK9 but not to human PCSK7, human MBTPSI (PCSK8) or human CD86 (data not shown).

Example 19 Blocking Human PCSK9 Binding to LDL Receptor with GLP-1 Analogue Peptide in Fusion with Anti-PCSK9 Antibody

Ability of the GLP-1 analogue anti-PCSK9 fusion HS9_DSB7_V19A to block the binding of human PCSK9 to human LDL receptor was assessed using an ELISA competition assay. Anti-PCSK9 antibody HS9 and irrelevant isotype match NIP228 human IgG1-TM were used as positive and negative controls respectively.

Binding of biotinylated human PCSK9 (in house) at 5 ug/mL in 1× Phosphate Buffered Saline, 3% skimmed milk to human LDL-R (R&D Systems) coated overnight at 10 ug/mL onto 96 well MaxiSorb plate (NUNC) was detected by ELISA using cryptate labelled streptavidin (Perkin Elmer) diluted at 100 ng/mL in Delfia Buffer (Perkin Elmer). That interaction was challenged using a 3-fold serial dilution, starting at 100 ug/mL, of compounds co-incubated for 2 h at room temperature with the biotinylated PCSK9 reagent in the LDL receptor coated wells. Fluorescence signal was read on the Perkin Elmer Envision machine using a 340 nm excitation and 620 nm emission. Percentage of specific binding was calculated by subtracting the background signal obtained with no LDL receptor coated onto the plate normalized with the maximum specific binding signal obtained with no competitor compound minus background level.

Biochemical inhibition of PCSK9 binding to LDL receptor is presented in FIG. 25.

Fusion molecule HS9_DSB7_V19A can block the binding of biotinylated human PCSK9 to recombinant LDL receptor with similar IC₅₀ compared to the positive control anti-PCSK9 antibody HS9 (4.3E-8 and 3.5E-8 M respectively).

Example 20 LDL Uptake by HEPG2 Cells Treated with GLP-1 Analogue Peptide in Fusion with Anti-PCSK9 Antibody

Ability of the GLP-1 analogue anti-PCSK9 fusion HS9_DSB7_V19A to block PCSK9 activity and restore LDL uptake was tested in HepG2 hepatic cells as followed. Anti-PCSK9 antibody PC9#2 and irrelevant isotype match NIP228 human IgG1-TM were used as positive and negative controls respectively.

Human HepG2 cells were seeded in black, clear bottom 96-well Greiner plates at a concentration of 2×10⁴ cells per well in DMEM medium (Gibco) supplemented with 10% lipoprotein deficient serum (Sigma) and incubated at 37° C. (5% CO₂) overnight. To complex PCSK9 with the tested compound, 45 nM of human Avi_PCSK9_FLAG_His (in house) was incubated with or without the tested compound at various concentrations in DMEM +10% LPDS for 1 hour at room temperature. All media was removed from the cell plate and the PCSK9/compound mixtures were transferred to the plate and incubated for 1 hour. Bodipy-LDL (Molecular Probes), diluted in DMEM+10% LPDS to a final concentration of 50 nM, was next transferred to the cells and the plate incubated for 5 hours at 37° C. (5% CO₂). Cells were washed thoroughly with PBS, stained with the nuclear dye Hoescht and fixed using formaldehyde at a final concentration of 3.7% (v/v). Assay plates were read for cell-associated fluorescence using the Cellomics ArrayScan VTi high content imaging system. Hoescht staining was measured in channel 1 using the BGRFR_386_(—23) filter and Bodipy-LDL in channel 2 using the BGRFR_485_20 filter. Images were analysed using the Compartmental Analysis v4 algorithm.

Inhibition of PCSK9-dependent loss of LDL uptake by HepG2 cells is presented in FIG. 26.

Fusion molecule HS9_DSB7_V19A can restore LDL-uptake by HepG2 cells treated with human PCSK9 with similar IC₅₀ compared to the positive control anti-PCSK9 antibody PC9#2 (2.5E-8 and 3.1E-8 M respectively).

Example 21 Potency at GLP-1 Receptors Across Species of GLP-1 Analogue Peptide in Fusion with Anti-PCSK9 Antibody

Cross-reactivity of the GLP-1 analogue anti-PCSK9 fusion HS9_DSB7_V19A at human, cynomolgus, mouse and rat GLP-1 receptors was tested in a cAMP production assay as previously described by using stable cell lines overexpressing the receptor of interest. GLP1-Fc fusion (in house) and GLP-1 peptide were used as positive controls.

Potency at the different GLP-1 receptors is summarised in Table 23.

TABLE 23 Potency of lead molecule HS9_DSB7_V19A at GLP-1 receptor across species Potency at GLP-1R in cAMP assay across species (M) # Compound human cynomolgus mouse rat 1 HS9_DSB7_V19A 3.73E−09 5.83-10 5.75-10 3.83E−11 2 GLP1-Fc(Gamma4) 7.72E−11 1.09E−11 8.98E−11 8.92E−12 3 GLP-1 peptide 1.61E−11 1.03E−11 2.69E−11 9.31E−11

Fusion molecule HS9_DSB7_V19A can activate GLP-1 receptor across all the four tested species.

Example 22 Several Compounds were Identified by Reducing Potency at the Human GLP-1 Receptor

To reduce DSB7 potency, certain residues in the peptide were mutated. Peptide Ab fusions to SEQ ID NO: 2 with linker SEQ ID NO: 4 at the desired potency, here shown in green triangles, were further analyzed for specificity at human GLP1-R and species cross reactivity. See FIG. 27.

The variants are shown as HS9_DSB7_G2V (Gly₂->Val) (SEQ ID NO: 44 and SEQ ID NO: 1); HS9_DSB7_E15A (Glu₁₅->Ala) (SEQ ID NO: 45 and SEQ ID NO: 1); HS9_DSB7_V19A (Val19->Ala) (SEQ ID NO: 47 and SEQ ID NO: 1); HS9_DSB7_L26I (Leu₂₆->Ile) (SEQ ID NO: 46 and SEQ ID NO:1) (compared to Val₁₉->Ala (SEQ ID NO: 3)). These four variants were selected for final characterization. Results are shown in FIG. 27.

Example 23 Fusion Molecule with Reduced Potency

The PCSK9/GLP-1 fusion molecule exhibits the desired potency on the GLP-1 receptor, as shown in FIG. 28A. The potency of this compound has been reduced to minimize nausea. Table 24 shows that HS9_DSB7_V19A has a 57.7 fold reduced potency with respect to dulaglutide. This engineered reduction of potency provides the desired effect of reducing nausea and other untoward effects.

TABLE 24 Fusion Molecule with Reduced Potency Mean Fold Change Over Max Activation Sample ID EC50 (M) Benchmark (%) HS9_DSB7_V19A 4.4E−09 57.7 98 (heavy chain of SEQ ID NO: 1 and light chain fusion of SEQ ID NO: 47) GLP-1 (SEQ ID NO: 1.6E−11 0.2 100 29) GLP1-Fc(G4) 7.7E−11 1.0 100 (dulaglutide)

PCSK9/GLP-1 fusion of Example 1 exhibits an Ab exposure/GLP-1 activity profile sufficient to enable weekly dosing, for example, as shown in FIG. 28B. pK stability study in rat of 58.5 mg/kg of HS9_DSB7_V19A injected into rat. Concentration of the fusion compound is measured in the serum over time. Samples taken from rat are analyzed for both activity of the test compound and concentration of the test compound in serum. Data from the GLP-1 activity portion is used to back calculate for concentration of “active” compound. The line with closed circles is the concentration of HS9_DSB_V19A and line with closed squares is the concentration of active HS9_DSB_7_V19A (i.e., having GLP-1 activity) for the same samples.

Example 24 Specificity for GLP-1 Receptor Compared to Closely Related Human Receptors of GLP-1 Analogue Peptide in Fusion with Anti-PCSK9 Antibody

Specificity of the GLP-1 analogue anti-PCSK9 fusion HS9_DSB7_V19A for GLP-1 receptor compared to related human receptors was tested in a cAMP production assay as previously described by using stable cell lines overexpressing the receptor of interest: glucagon, GIP, GLP-2 and secretin receptors. Specific agonist peptides for each of the four receptors were used as positive controls.

Data are shown in FIGS. 29A-D (A: Glucagon receptor, B: GIP receptor, C: GLP-2 receptor, D: Secretin receptor).

Fusion molecule HS9_DSB7_V19A is specific for GLP-1 receptor and does not activate any of the four tested closely related receptors.

Example 25 Further Characterization of Fusion Molecule Demonstrates a Favorable In Vivo profile.

The PCSK9/GLP-1 fusion molecule of Example 2 (HS9_DSB7_V19A (heavy chain of SEQ ID NO: 1 and light chain fusion of SEQ ID NO: 47)) shows superior glucose control and weight loss over time, including at day 7 post dose. Data is shown against dulaglutide and PC9_2_VH and VL (SEQ ID NOS: 8 and 9). Animals were dosed at day 0 with the compounds and then their body weight was measured over time to determine a change in body weight.

The fusion molecule has shown that it binds purified PCSK9 with high affinity, it restores LDLc uptake in HEPG2 cells, stimulates GLP-1R at a desired potency, promotes weight loss and demonstrates favorable exposure/activity profile in rat PK to support weekly dosing and sustained GLP-1 activity in vivo.

Results are provided in FIGS. 30A-B.

Example 26 Impact of the Linker on the Activities of GLP-1 Analogue Peptide in Fusion With Anti-PCSK9 Antibody

The compound HS9_DSB7_V19A has a linker of SEQ ID NO: 4 corresponding to a Gly₄Ser motif repeated three times between the peptide moiety and the antibody light chain. To investigate the impact of the linker length between the GLP-1 analogue peptide DSB7_V19A (SEQ ID NO: 3) when fused to the light chain of anti-PCSK9 antibody HS9 (light chain: SEQ ID NO: 2 and heavy chain: SEQ ID NO: 1), three fusions with a reduced linker length were generated: HS9_DSB7_V19A_L2 (SEQ ID NO: 419) having a linker corresponding to the Gly₄Ser motif repeat two times (linker: SEQ ID NO: 403), HS9_DSB7_V19A_L1 (SEQ ID NO: 420) having a linker corresponding to the Gly₄Ser motif repeat one time (SEQ ID NO. 27) and HS9_DSB7_V19A_L0 with no linker between the peptide and the antibody light chain (SEQ ID NO: 421).

Compounds were tested for both binding to recombinant human PCSK9 by ELISA and activity at the human GLP-1 receptor using the cAMP assay cell based assay as described in Example 4.

Binding ELISA was performed by coating human PCSK9 (in house) at 10 ug/mL in 1× Phosphate Buffered Saline. After plate blocking with 1× Phosphate Buffered Saline, 3% skimmed milk, compounds were added at 100 ug/mL in PBS and incubated for 2 h at room temperature before washing. Bound compounds were detected using cryptate labelled Fc specific anti human IgG (Perkin Elmer) diluted at 100 ng/mL in Delfia Buffer (Perkin Elmer). Fluorescence signal was read on the Perkin Elmer Envision machine using a 340 nm excitation and 620 nm emission. HS9_DSB7_V19A was used as positive control and irrelevant isotype match NIP228 used to determine the background level.

PCSK9 binding and GLP-1 receptor activation data are shown in FIGS. 31 and 32, respectively.

All additional fusion molecules are able to bind human PCSK9 to a similar level compared to HS9_DSB7_V19A. Tested fusion molecules can also activate human GLP-1 receptor in the cAMP cell based assay but reducing linker length is having a negative impact on compound potency. In that assay, EC₅₀ for HS9_DSB7_V19A, HS9_DSB7_V19A_L2, HS9_DSB7_V19A_L1 and HS9_DSB7_V19A_L0 are 5.0 nM, 15.6 nM, 68.7 nM and 537 nM, respectively.

Example 27 In Vitro Characterization of Stable GLP-1 Analogue Peptide in Fusion with Anti-PCSK9 Antibodies

The GLP-1 analogue peptide DSB7_V19A of SEQ ID NO: 3) was fused using a linker of SEQ ID NO:4 to the light chain of other anti-PCSK9 antibodies than HS9:

1_ PC9#1 with antibody variable heavy chain of SEQ ID NO:10 and antibody variable light chain of SEQ ID NO:11.

2_ PC9#3 with antibody variable heavy chain of SEQ ID NO:404 and antibody variable light chain of SEQ ID NO:405.

3_ PC9#4 with antibody variable heavy chain of SEQ ID NO:406 and antibody variable light chain of SEQ ID NO:407.

4_ PC9#5 with antibody variable heavy chain of SEQ ID NO:408 and antibody variable light chain of SEQ ID NO:409.

5_ PC9#6 with antibody variable heavy chain of SEQ ID NO:410 and antibody variable light chain of SEQ ID NO:411.

6_ PC9#7 with antibody variable heavy chain of SEQ ID NO:412 and antibody variable light chain of SEQ ID NO:413.

Fusions were tested for both binding to recombinant human PCSK9 by ELISA as described in Example 26 and activity at the human GLP-1 receptor using the cAMP cell based assay as described in Example 4. HS9_DSB7_V19A and NIP228 isotype match were used as positive and negative controls respectively.

PCSK9 binding and GLP-1 receptor activation data are shown in FIGS. 33 and 34, respectively.

All seven compounds tested in the human GLP-1 receptor cAMP assay are able to activate the receptor. Potency among the panel is ranging from 1 to 350 nM for PC9#6_DSB7_V19A and PC9#5_DSB7_V19A, respectively. HS9_DSB7_V19A has a potency of 5 nM in that assay.

In addition, all tested fusions are also able to bind strongly to human PCSK9 by ELISA at the exception of PC9#7_DSB7_V19A which binds poorly and PC9#6_DSB7_V19A which does not bind.

Example 28 In Vitro Characterisation of Stable GLP-1 Analogue Peptide in Fusion with an Anti-B7-H1 Antibody

The GLP-1 analogue peptide DSB7_V19A of SEQ ID NO: 3 was fused using a linker of SEQ ID NO:4 to the light chain of the anti-B7-H1 antibody 2.7A4 described in patent WO2011066389. Anti B7-H1 antibody 2.7A4 has a variable heavy chain of SEQ ID NO:422 and a variable light chain of SEQ ID NO:423.

Fusion was tested for both binding to recombinant human B7-H1 by ELISA and activity at the human GLP-1 receptor using the cAMP assay cell based assay as described in Example 4.

Binding ELISA was performed by coating human B7-H1 (in house) at 5 ug/mL in 1× Phosphate Buffered Saline. After plate blocking with 1×Phosphate Buffered Saline, 3% skimmed milk, compounds were added at 10 ug/mL in PBS and incubated for 2 h at room temperature before washing. Bound compounds were detected using cryptate labelled Fc specific anti human IgG (Perkin Elmer) as described in Example 25. Anti-B7-H1 antibody 2.7A4 and irrelevant isotype match NIP228 were used as positive and negative control respectively.

B7-H1 binding and GLP-1 receptor activation data are shown in FIGS. 35 and 36, respectively.

Fusion 2.7A4 DSB7 V19A is able to bind human B7-H1 to a similar level than the positive control 2.7A4 antibody. The fusion can also activate human GLP-1 receptor in the cAMP cell based assay with a potency of 100 nM compared to 5 nM for HS9_DSB7_V19A.

Example 29 Pharmacokinetics and Pharmacodynamics in Rat of GLP-1 Analogue Peptide in Fusion with Anti-PCSK9 Antibody Following Single Intravenous Dose

A PKPD study for the peptide antibody fusion HS9_DSB7_V19A following a single intravenous bolus in CD rats was conducted in order to assess its in vivo stability, by measuring both compound exposure and concentration in active GLP-1, as well as target engagement by measuring concentration in free rat PCSK9.

Fusion molecule was injected at 10, 30 and 60 mg/kg. Anti-PCSK9 mAb HS9, without a peptide attached to it, was used as control and injected at 60 mg/kg. Blood samples were collected at:

1: Pre-dose—0.5 h—6 h—24 h—48 h—96 h and 168 h for the 10 mg/kg treatment group;

2: Pre-dose—0.5 h—24 h—48 h—96 h—168 h and 240 h for the 30 mg/kg treatment group; and

3: Pre-dose—0.5 h—24 h—72 h—168 h—336 h and 504 h for the 60 mg/kg treatment groups.

Concentrations of total human IgG1 antibody (exposure) and of active GLP-1 compound in rat serum samples were quantified as described in Example 4.

HS9_DSB7_V19A concentration over time in total and active GLP-1 compound in rat serum for the three tested doses (10, 30 and 60 mg/kg) are shown in FIG. 37.

Area under curve (AUC) for both total compound and active GLP-1 are summarised in Table 25. Calculating the ratio between active GLP-1 and total compound AUC is one way to evaluate in vivo stability. A ratio of one is corresponding to a fully stable compound in the tested conditions.

Data for the parent fusion molecule PC9#2_Exe4 comprising Exendin-4 in light chain fusion with the anti-PCSK9 mAb PC9#2 (See Example 4) have also been included for comparison.

TABLE 25 Total and active GLP-1 AUC0-t following a single IV injection of the fusion molecule HS9_DSB7_V19A compared to the parent fusion molecule PC9#2_Exe4 AUC0-t Active/ AUC0-t Active Total Total GLP-1 AUC Compound Design (day · nmol) (day · nmol) Ratio HS9_DSB7_V19A 10 mg/kg IV 1595 1216 0.76 HS9_DSB7_V19A 30 mg/kg IV 8702 6230 0.72 HS9_DSB7_V19A 60 mg/kg IV 24535 26494 1.08 PC9#2_Exe4  1 mg/kg IV 299 40 0.13

HS9_DSB7_V19A displays a greater Active/Total AUC ratio compared to the parent molecule PC9#2_Exe4 demonstrating an improved in vivo stability profile in rat for activity at GLP-1 receptor.

Determination of free rat PCSK9 concentration in serum samples was based on a sandwich ligand binding assay method using the MSD® platform. Free rat PCSK9 was captured using the anti-PCSK9 mAb HS9 that was first non-specifically adsorbed on to the carbon surface of a Standard Bind MSD® plate. Anti-PCSK9 antibody from Abcam (Product Number ab125251) was labelled in-house with MSD® SULFO-TAG™ and used as detection reagent. Plate was then read using the MSD® Sector Imager 6000 (SI6000) instrument.

Engagement of HS9_DSB7_V19A to rat PCSK9 over time was evaluated by measuring the concentration of free antigen in the rat serum samples. Data for the three dosing groups (10, 30 and 60 mg/kg) are shown in Table 26 and FIG. 38.

TABLE 26 Concentration of Free Antigen in Rat Plasma Samples Over Time 10 mg/kg IV 30 mg/kg IV 60 mg/kg IV Mean free Mean free Mean free rat rat rat [PCSK9] in [PCSK9] in Time [PCSK9] in Time (h) ng/mL Time (h) ng/mL (h) ng/mL 0 837.2 0 741.8 0 688.5 0.5 below 0.5 125.8 0.5 below LLOQ LLOQ 6 26.7 24 below 24 below LLOQ LLOQ 24 74.1 48 below 72 below LLOQ LLOQ 48 162.5 96 below 168 below LLOQ LLOQ 96 301.0 168  70.9 336  45.4 168 374.7 240 130.3 504 135.8

HS9_DSB7_V19A is able to suppress rat PCSK9 below 90% at all tested doses but the duration of suppression is dose dependent. Free rat PCSK9 is again detectable at 6 h, 168 h and 336 h after injection when HS9_DSB7_V19A is dosed at 10, 30 and 60 mg/kg respectively.

Example 30 Pharmacokinetics and Pharmacodynamics in Rat of GLP-1 Analogue Peptide in Fusion with Anti-PCSK9 Antibody Following Single Subcutaneous Dose

A pharmacokinetic study for the GLP-1 analogue peptide antibody fusion HS9_DSB7_V19A following a single subcutaneous bolus at 60 mg/kg in CD rats was performed in order to compare routes of administration and their potential impact on compound exposure and in vivo stability.

Fusion molecule was injected at 40 mg/mL using a 1.5 mL/kg regimen. Blood samples were collected at Pre-dose—6 h—24 h—48 h—96 h—168 h and 240 h.

Concentrations of total human IgG1 antibody and of active GLP-1 compound as well as concentrations in free rat PCSK9 antigen in the serum samples were determined as described in Example 4 and Example 29.

Total and active compound for HS9_DSB7_V19A and free rat PCSK9 concentrations are shown in FIG. 39. A maximum compound concentration of around 1000 nM was observed between 48 h and 96 h post-injection. Concentration in free rat PCSK9 is dropping sharply below the lower limit of quantification of the assay after compound injection and can be detected again at 168 h.

Area under curve (AUC) for both total and active compound were calculated and compared (Table 27) to model prediction using the data from the single intravenous dose injection described in Example 29. Fraction of absorption and absorption rate were set up to 75% and 0.3 d¹ respectively.

TABLE 27 Calculated and predicted Area Under Curve following a single SC injection of the fusion molecule HS9_DSB7_V19A SC data Predicted using 60 mg/kg IV data Exposure AUC0-t (day nmol) 7815 8103 Active GLP-1 AUC0-t (day 5796 5739 nmol) Active/Exposure AUC ratio 0.74 0.71

Exposure and active GLP-1 AUC of HS9_DSB7_V19A after a single subcutaneous injection at 60 mg/kg are similar to those predicted using the single intravenous injection data. This demonstrates that a subcutaneous route of injection has no significant impact on in vivo compound stability compared to an intravenous route of administration.

Example 31 Rodent Pharmacology-Antidiabetic Effects of a GLP-1 Analogue Peptide in Fusion with an Anti-PCSK9 Antibody

In order to confirm that the engineered nature of the GLP-1 analogue peptide portion of the HS9_DSB7_V19A fusion molecule retained antidiabetic activity in vivo, several; rodent pharmacology studies in normal, obese, and diabetic mouse models were performed.

A) Acute and Semi-Acute Effects of HS9_DSB7_V19A on Glucose Tolerance in C57B16 Mice

A single dose of HS9_DSB7_V19A was administered subcutaneously in normal C57B16 mice at either 1 or 50 mg/kg. The efficacy of the GLP-1 analogue component of the fusion molecule was evaluated by multiple glucose challenges (oral glucose tolerance test) at 4, 48, and 168 hours post administration of HS9_DSB7_V19A. Anti-PCSK9 mAb HS9 without a GLP-1 analogue peptide fused to it was administered at 50 mg/kg as a negative control for glucose tolerance, while Liraglutide (Victoza) and a GLP-1 analogue-Fcγ4 fusion (similar to Dulaglutide) were administered at 0.2 and 1 mg/kg, respectively. Due to the short half-life of Liraglutide, this compound was administered 2 hours prior to each glucose challenge while the GLP-1 analogue-Fcγ4 fusion molecule was administered once, at the same time as the HS9_DSB7_V19A test compound.

30 male C57B16 mice from Taconic Denmark were acclimatized for five days before experimentation. On day −1 of the study, animals were randomized into 5 groups based on body weight.

The experimental groups were as follows:

Group 1: Anti-PCSK9 mAb HS9 without GLP-1 analogue peptide component (negative control) −50 mg/kg subcutaneous dose

Group 2: Liraglutide (positive control) −0.2 mg/kg subcutaneous dose

Group 3: HS9_DSB7_V19A −1 mg/kg subcutaneous dose

Group 4: HS9_DSB7_V19A −50 mg/kg subcutaneous dose

Group 5: GLP-1 analogue-Fcγ4 fusion −1 mg/kg subcutaneous dose

In order to assess efficacy of the GLP-1 analogue component of the fusion molecule over an extended period of time post dosing, 3 Oral Glucose Tolerance Tests (OGTT) were performed at days 0, 2 and 7. Animals were fasted for 4 hours prior to the oral glucose challenge(s). Both doses of HS9_DSB7_V19A, inactive control and GLP-1 analogue-Fcγ4 fusion were administered once, 4 hours prior to the Day 0 OGTT. Liraglutide (positive control) was administered 2 hours prior to each glucose challenge at days 0, day 2 and day 7. At t=0 mice all mice are challenged with an oral glucose load of 2 g/kg glucose. Blood glucose is measured at t=−240, −120 and 0 minutes to establish a baseline and at t=15, 30, 60 and 120 minutes to monitor effects on glucose excursion. The results of the day 0, 2 and 7 OGTTs are presented in FIGS. 40A-C (day 0 (A), day 2 (B), day 7 (C)).

This study confirms the ability of a single, subcutaneous administration of HS9_DSB7_V19A at 50 mg/kg to improve glucose tolerance in normal C57B16 mice for at least 7 days.

As an additional measure of efficacy of the GLP-1 analogue component of the HS9_DSB7_V19A fusion molecule body weights of all were recorded once daily from day -3 to the end of the study. A single, subcutaneous administration of HS9_DSB7_V19A at 50 mg/kg induced a transient reduction in body weight at days 1 and 2. Percent body-weight change over time is shown in FIG. 41.

B) Acute and semi-acute effects of HS9_DSB7_V19A on glucose tolerance in C57B16 mice-dose response

In order to examine a dose response effect and to determine a maximally efficacious dose of HS9_DSB7_V19A on glucose tolerance and body weight reduction in normal C57B16 mice, a study similar in design to that described above was conducted with the following experimental groups and doses:

Group 1: Anti-PCSK9 mAb HS9 without GLP-1 analogue peptide component (negative control)-50 mg/kg subcutaneous dose

Group 2: Liraglutide (positive control) −0.2 mg/kg subcutaneous dose

Group 3: HS9_DSB7_V19A −10 mg/kg subcutaneous dose

Group 4: HS9_DSB7_V19A −30 mg/kg subcutaneous dose

Group 5: HS9_DSB7_V19A −60 mg/kg subcutaneous dose

Group 6: GLP-1 analogue-Fcγ4 fusion-1 mg/kg subcutaneous dose

On day −1 animals were randomized into these 6 groups based on body weight (n=6 animals per group). As previously, OGTTs were performed on Days 0, 2 and 7 and blood glucose was measured at t=−240, −120, 0, 15, 30, 60 and 120 minutes.

All three doses of HS9_DSB7_V19A resulted in similar levels of improved glucose tolerance at all three time points at which OGTTs were performed. FIGS. 42A-C illustrate the results from this oral glucose tolerance tests (day 0 (A), day 2 (B), day 7 (C)).

In contrast to the lack of a dose response observed for improvements in glucose homeostasis, a clear dose-dependent effect on body weight reduction was observed in this study. FIG. 43.

In this experimental model a single 10 mg/kg dose of HS9_DSB7_V19A generated a maximally efficacious level of improvement in glucose homeostasis in an OGTT performed 7 days post administration while the same dose did not have a statistically significant effect on body weight reduction at any point during the study.

C) Chronic Metabolic Effects of HS9_DSB7_V19A in a Diabetic db/db Mouse Model

In order to confirm the chronic vivo efficacy of HS9_DSB7_V19A on several metabolic parameters in a diabetic model, db/db (leptin receptor deficient) mice were utilized to examine the effects of weekly administration of HS9_DSB7_V19A on fasting glucose, glucose tolerance, body weight reduction and body mass composition.

In this study we examined the chronic metabolic effects of HS9_DSB7_V19A upon weekly dosing via a subcutaneous route. The vehicle control group and the positive control GLP-1 analogue-Fcγ4 fusion molecule were subcutaneously dosed twice weekly. In order to match the number of dosing manipulations to all animals in the study, the groups receiveing a weekly dose of HS9_DSB7_V19A were dosed with vehicle on the days the other animals received their second weekly dose of either vehicle (negative control) or positive control GLP-1 analogue-Fcγ4 fusion molecule.

60 male db/db mice from Charles River, Italy were primarily randomized on body weight and glycosylated hemoglobin (HbA1c) and secondarily on 4 hour fasting blood glucose. These animals were randomized into 5 groups (n=12) as follows:

Group 1: Vehicle control group (Phosphate Buffered Saline)—twice weekly (BIW) subcutaneous dosing

Group 2: GLP-1 analogue-Fcγ4 fusion (positive control) −1 mg/kg subcutaneous dose—twice weekly subcutaneous dosing

Group 3: HS9_DSB7_V19A −30 mg/kg subcutaneous dose—once weekly (QW)

Group 4: HS9_DSB7_V19A −10 mg/kg subcutaneous dose—once weekly (QW)

Group 5: HS9_DSB7_V19A −3 mg/kg subcutaneous dose—once weekly (QW)

The chronic study was run for 28 days post initial dose. Last dose of the BIW groups was on study day 24 whereas the last dose of the QW groups was on study day 21. The major efficacy endpoints of this study are body weight, fasting blood glucose, and glucose tolerance.

Body weight was measured three times weekly during the treatment period.

4 hour fasting blood glucose was measured once weekly at approximately 24 hours post dosing.

Glucose tolerance was measured on study day 22 at approximately 24 hours post dosing by Intraperitoneal glucose tolerance test (IPGTT). Animals were fasted for 4 hours before administration of a 1g/kg glucose bolus. Blood glucose was measured at t=0, 15, 30, 60, 120, and 180 minutes.

Body weight reduction in this study was not significant with the exception of one time point in the positive control GLP-1 analogue-Fcγ4 fusion molecule group. All three doses of HS9_DSB7_V19A did not result in a significant body weight reduction at any time during the course of the 28 day study. FIG. 44.

Weekly HS9_DSB7_V19A exhibited a dose dependent reduction in 4 hour fasting blood glucose as compared to vehicle control. FIG. 45.

Weekly dosed HS9_DSB7_V19A exhibited a dose dependent improvement in glucose tolerance as assessed by IPGTT at study day 22. FIG. 46.

D) Acute effect of single dose HS9_DSB7_V19A on glucose tolerance in a Diet-Induced Obesity (DIO) mouse model

A single dose of HS9_DSB7_V19A was administered subcutaneously in Diet-induced obese (DIO) mice at 0.1, 1 or 10 mg/kg. The efficacy of the GLP-1 analogue component of the fusion molecule was evaluated by separate glucose challenges (intraperitoneal glucose tolerance test) at 4 and 168 hours post administration of HS9_DSB7_V19A. Anti-PCSK9 mAb HS9 without a GLP-1 analogue peptide fused to it was administered once at 10 mg/kg as a negative control for effects on glucose tolerance. As a positive control, the GLP-1 analogue-Fcγ4 fusion (similar to Dulaglutide) used in the previous experiments was administered at 1 mg/kg twice weekly (BIW). In order to simulate the dosing regimen of the positive control GLP-1 analogue-Fcγ4 fusion, all animals in the negative control group and the HS9_DSB7_V19A experimental groups were dosed with vehicle BIW. The study duration was for 21 days following the first dose. Primary endpoint was effects on glucose tolerance in IPGTT on day 0 (4 hours post dose) and Day 7. Secondary endpoint was body weight reduction.

50 Male, 21 week old DIO mice on 60% high fat diet for 15 weeks prior to study start were obtained from Jackson labs (JAX: 380050). Just prior to study start, animals were randomized into 5 groups based on body weight.

The experimental groups (n=10 per group) were as follows:

Group 1: Anti-PCSK9 mAb HS9 without GLP-1 analogue peptide component (negative control) −10 mg/kg subcutaneous dose—single dose

Group 2: HS9_DSB7_V19A −0.1 mg/kg subcutaneous dose—single dose

Group 3: HS9_DSB7_V19A −1 mg/kg subcutaneous dose—single dose

Group 4: HS9_DSB7_V19A −10 mg/kg subcutaneous dose—single dose

Group 5: GLP-1 analogue-Fcγ4 fusion −1 mg/kg subcutaneous dose—twice weekly dosing (BIW)

In order to assess efficacy of the GLP-1 analogue component of the fusion molecule over an extended period of time post dosing, two Intraperitoneal Glucose Tolerance Tests (IPGTT) were performed at days 0 and 7. Animals were fasted for 6 hours prior to the IP glucose challenge(s). All three doses of HS9_DSB7_V19A, inactive control and GLP-1 analogue-Fcγ4 fusion were administered once, 4 hours prior to the Day 0 OGTT. The GLP-1 analogue-Fcγ4 fusion was administered on days 0, 3, 7, 10, 14 17 and 21. On day 0 all groups were dosed with test compounds 4 hours prior to the IP glucose challenge while on day 7 the GLP-1 analogue-Fcγ4 fusion was administered 4 hours prior to the IP glucose challenge while all other groups were dosed with vehicle only . For each day in which IPGTTs were performed, at t=0 mice all mice were challenged with an IP glucose load of 1.5 g/kg glucose. Blood glucose was measured at t=−240 and 0 minutes to establish a baseline and at t=15, 30, 45, 60, 90 and 120 minutes to monitor effects on glucose excursion.

The results of the day 0 and 7 IPGTTs are presented in FIGS. 47A-B.

In order to assess effects of the GLP-1 component of HS9_DSB7_V19A on body weight, all animals were weighed daily for the entire course of the study. Effects on body weight are presented in 48A-B.

E) Effects of multiple doses of HS9_DSB7_V19A on body weight in DIO mice.

In order to establish a dose-response in body weight reduction HS9_DSB7_V19A was administered subcutaneously to diet-induced obese mice at 3, 10 and 30 mg/kg once weekly (QW). The duration of the study was 28 days and primary endpoint was body weight reduction. Secondary evaluation of glycemic parameters included analyses included fed glucose throughout the course of the study and measurements of terminal fasting glucose. As in our previous studies in both DIO and db/db mice, the GLP-1 analogue-Fcγ4 fusion was administered subcutaneously, twice weekly (BIW) at 1 mg/kg as a positive control for both glucose control and body weight loss.

Male, 21 week old DIO mice on 60% high fat diet for 15 weeks prior to study start were obtained from Jackson labs (JAX: 380050). Just prior to study start, animals were randomized into 5 groups based on body weight (n=8 animals per group).

The experimental groups (n=8 per group) were as follows:

Group 1: Vehicle control

Group 2: HS9_DSB7_V19A −3 mg/kg subcutaneous dose—once weekly (QW)

Group 3: HS9_DSB7_V19A −10 mg/kg subcutaneous dose—once weekly (QW)

Group 4: HS9_DSB7_V19A −30 mg/kg subcutaneous dose—once weekly (QW)

Group 5: GLP-1 analogue-Fcγ4 fusion −1 mg/kg subcutaneous dose—twice weekly dosing (BIW)

In order to assess effects of the GLP-1 component of HS9_DSB7_V19A on body weight (the primary endpoint of this study), all animals were weighed daily for the entire course of the study. Effects on body weight are presented in FIG. 49.

As a secondary endpoint and in order to assess effects of the GLP-1 component of HS9_DSB7_V19A on glycemic control in a weekly dose setting, fed glucose was measured at days 0, 7, 11, 14, 21 and 26 and fasting glucose was measured at just prior to study termination (day 28). Results are presented in FIGS. 50A-B.

EQUIVALENTS

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiments may be practiced in many ways and the claims include any equivalents thereof. 

What is claimed is:
 1. A dual active fusion molecule for the treatment of diabetes comprising an anti-PCSK9 antibody stably fused to a GLP-1 peptide, wherein the anti-PCSK9 antibody binds a PCSK9 polypeptide and the GLP-1 peptide binds a GLP-1 receptor.
 2. The fusion molecule of claim 1 wherein the GLP-1 peptide is fused to the PCSK9 antibody via a linker peptide.
 3. The fusion molecule of claim 2 wherein the linker peptide is fused to the C-terminus of the GLP-1 peptide.
 4. The fusion molecule of any one of claims 1-3, wherein the GLP-1 peptide comprises the amino acid sequence of SEQ ID NO:
 36. 5. The fusion molecule of any one of claims 1-3, wherein the GLP-1 peptide comprises the amino acid sequence of SEQ ID NO:
 3. 6. The fusion molecule of claim 5 wherein the Cys18 of the GLP-1 peptide forms a disulfide bridge with the C terminus of the GLP-1 peptide.
 7. The fusion molecule of any one of claims 1-6, wherein said fusion molecule controls glucose and/or reduces LDL in an animal.
 8. The fusion molecule of claim 7, wherein the animal is a human.
 9. A dual active fusion molecule for the treatment of diabetes comprising an anti-PCSK9 antibody stably fused to a GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 3, wherein the C-terminus of the GLP-1 peptide is fused via a peptide linker to the anti-PCSK9 antibody, and wherein the anti-PCSK9 antibody binds a PCSK9 polypeptide and the GLP-1 peptide binds a GLP-1 receptor.
 10. The dual active fusion molecule of claim 9, wherein the GLP-1 peptide is fused via a peptide linker to the light chain of the anti-PCSK9 antibody.
 11. The dual active fusion molecule of claim 9, wherein the GLP-1 peptide is fused via a peptide linker to the heavy chain of the anti-PCSK9 antibody.
 12. A dual active fusion molecule comprising an antibody stably fused to a GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 3, wherein the C-terminus of the GLP-1 peptide is fused via a peptide linker to the antibody, and wherein the antibody binds a target polypeptide and the GLP-1 peptide binds a GLP-1 receptor.
 13. The dual active fusion molecule of claim 12, wherein the antibody is an anti-PCSK9 antibody.
 14. The dual active fusion molecule of claim 13, wherein the GLP-1 peptide is fused via a peptide linker to the light chain of the anti-PCSK9 antibody.
 15. The dual active fusion molecule of claim 13, wherein the GLP-1 peptide is fused via a peptide linker to the heavy chain of the anti-PCSK9 antibody.
 16. The dual active fusion molecule of claim 10 or 14, wherein the light chain of the anti-PCSK9 antibody is at least 90% identical to the amino acid sequence of SEQ ID NO:
 2. 17. The dual active fusion molecule of claim 16, wherein the light chain of the anti-PCSK9 antibody comprises the amino acid sequence of SEQ ID NO:
 2. 18. The dual active fusion molecule of claim 11 or 15, wherein the heavy chain of the anti-PCSK9 antibody is at least 90% identical to the amino acid sequence of SEQ ID NO:
 1. 19. The dual active fusion molecule of claim 18, wherein the heavy chain of the anti-PCSK9 antibody comprises the amino acid sequence of SEQ ID NO:
 1. 20. The dual active fusion molecule of any one of claims 12-19 wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:
 4. 21. The dual active fusion molecule of any one of claims 12-20, wherein the dual active fusion molecule is for the treatment of diabetes.
 22. The dual active fusion molecule of any one of claims 12-21, wherein the Cys18 of the GLP-1 peptide forms a disulfide bridge with the C terminus of the GLP-1 peptide.
 23. The dual active fusion molecule of any one of claims 12-22, wherein the dual active fusion molecule controls glucose and/or reduces LDL in an animal.
 24. The dual active fusion molecule of claim 23, wherein the animal is a human.
 25. A dual active fusion molecule for the treatment of diabetes comprising an anti-PCSK9 antibody stably fused to a GLP-1 peptide that has reduced potency at the human GLP-1 receptor compared to a GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29, wherein the C-terminus of the GLP-1 peptide is fused via a peptide linker to the anti-PCSK9 antibody, and wherein the anti-PCSK9 antibody binds a PCSK9 polypeptide and the GLP-1 peptide binds a GLP-1 receptor.
 26. The dual active fusion molecule of claim 25, wherein the GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29 is fused using a linker comprising SEQ ID NO:4 to the amino acid sequence comprising SEQ ID NO:
 416. 27. The dual active fusion molecule of claim 25 or 26, wherein the potency at the human GLP-1 receptor is reduced by 30 to 60 fold compared to a GLP-1 peptide comprising the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO:
 29. 28. A method of treating Type 2 Diabetes comprising administering to a subject in need thereof, a fusion molecule of any one of claims 1-27.
 29. A method of controlling glucose in a subject comprising administering to a subject in need thereof, a fusion molecule of any one of claims 1-27.
 30. A method of reducing low density lipoprotein (LDL) in a subject comprising administering to a subject in need thereof, a fusion molecule of any one of claims 1-27.
 31. A method of controlling glucose and reducing LDL in a subject comprising administering to a subject in need thereof, a fusion molecule of any one of claims 1-27.
 32. A method of promoting weight loss, controlling glucose and reducing LDL in a subject comprising administering to a subject in need thereof, a fusion molecule of any one of claims 1-27.
 33. The method of any one of claims 28-32, wherein the subject has Type 2 diabetes.
 34. The method of any one of claims 28-32, wherein the subject has metabolic syndrome.
 35. An isolated polynucleotide encoding the fusion molecule of any one of claims to
 27. 36. A vector comprising the polynucleotide of claim
 35. 37. A host cell comprising the polynucleotide of claim 35 or the vector of claim
 36. 38. A method of making the fusion molecule of any one of claims to 27, comprising culturing the host cell of claim 35 under conditions allowing expression of the fusion molecule, and recovering the fusion molecule.
 39. A pharmaceutical composition comprising the fusion molecule of any one of claims 1 to 27, and a carrier.
 40. A kit comprising the composition of claim
 39. 